First Report of Neofabraea actinidiae causing a Cranberry Fruit Rot in Oregon.

Cranberry (Vaccinium macrocarpon, L.) is a commercial small fruit that is native to North America. Oregon ranks fourth in cranberry production in the U.S.A. with 1052 Ha of cranberry beds and annual production of 23,590 metric tonnes (USDA NASS 2021). Cranberry fruit rot diseases are caused by a complex of 15 fungal pathogens belonging to 10 genera (Polashock et al. 2017). In fruit rot surveys of 'Stevens' cranberry beds in Coos and Curry Counties, Oregon, berries were collected before harvest and sorted by symptoms (e.g. softening, shriveling, or discoloration). Cranberries with field rot symptoms were surface-disinfested and lesion margin tissue placed on V8 agar. Cultures were incubated at room temperature and outgrowing fungi were transferred twice onto fresh V8 agar to obtain single isolates. Storage rots that developed on asymptomatic cranberries held at 4°C for 8 weeks were processed similarly. Since 2017, we periodically isolated Neofabraea actinidiae, which is not a member of the cranberry fruit rot complex, from rotted cranberries (Polashock et al. 2017). In 2022, N. actinidiae was isolated from 22% of 45 cranberries collected from an organically managed farm and developed storage rot and from 6% of 50 storage-rot cranberries from three conventionally managed farms. Symptoms associated with N. actinidiae on 'Stevens' cranberries often include softening and brown tissue surrounded by a yellow-colored ring. On V8 agar, N. actinidiae grew as compact white circular colonies with dense aerial hyphae near the center and accompanied by a red pigment in the agar. Pink-colored mucoidal irregular conidiomata often developed on the colony after 3 weeks. Conidia were hyaline, aseptate, and ellipsoidal to fusiform ranging from 7.5 to 12.6 µm long X 3.5 to 5.6 µm wide (n=100). Genomic DNA was extracted from N. actinidiae isolates from cranberries in 2017 and 2022. ß-tubulin and the ITS 5/4 region were amplified and sequenced with primers Bt-T2m-Up/Bt-LEV-Lo1 and ITS5/ITS4 using conditions of de Jong et al. (2001) and White et al. (1990), respectively. Sequences were deposited in NCBI Genbank (Accessions: OR606303, OR606305, OR606306, & OR606309 for ITS; OR610314, OR610316, OR610317, & OR610320 for ß-tubulin). BlastN analysis of ß-tubulin (695 bp) and ITS (489-490 bp) had a 99.6 to 99.8% and 99.8 to 100% identity, respectively, to Neofabraea actinidiae (CBS 121403) (Accession numbers: KR859285 ß-tubulin and KR859079 ITS). Phylogenetic analysis of concatenated sequences using Tamura-Nei neighbor-joining (Tamura et al. 2004) confirmed identity of cranberry isolates as N. actinidiae. Koch's Postulates were confirmed with four N. actinidiae isolates from cranberry. Agar or hyphal plugs were placed in a 3 mm wound on the side of six surface-disinfested, asymptomatic berries and incubated in a moist chamber at 20°C for 15 days or 4°C for 55 days. Similar symptoms developed on each berry inoculated with N. actinidiae, but not agar alone. The fungus was recovered from symptomatic tissues and identity confirmed by colony morphology. N. actinidiae causes a ripe rot and storage rot in kiwifruit and is one of the species causing Bull's Eye Rot of pome fruits (Tyson et al. 2019). Cryptosporiopsis actinidiae (anamorph) was isolated from cranberries roots in British Columbia, CA, but considered unlikely to be the causal agent of dieback disease of cranberry vines (Sabaratnam et al. 2009). We have demonstrated that N. actinidiae causes a cranberry fruit rot in beds and in storage. Its prevalence, associated fruit rot symptoms, and disease incidence will continue to be monitored.

Taxonomic Reclassification of the Fungal Pathogen Causing Dry Berry Disease of Caneberries into the Division Ascomycota as Monilinia rubi.

As molecular genetic techniques improve and sequence data becomes available for more fungal species, taxonomic classifications historically based upon growth morphology alone are being revisited and occasionally reclassified. Herein, we present such an instance for the fungal pathogen that causes dry berry disease of caneberries. The organism was previously described as the basidiomycete fungus Rhizoctonia rubi based upon the pathogen's production of Rhizoctonia-like angular branching hyphae. Utilizing molecular genetic techniques unavailable when the pathogen was first characterized in 1959, three housekeeping gene regions (ITS, ß-tubulin, and G3PDH) were sequenced across 13 contemporary dry berry isolates, as well as the original 1959 R. rubi type strain, CBS382.59. The resulting neighbor-joining, maximum likelihood, and Bayesian phylogenies for single and multilocus sequences provide strong evidence that the dry berry pathogen was misclassified. This data, in addition to revisiting in vivo macroscopic and microscopic growth morphology, again comparing contemporary dry berry isolates to the CBS382.59 type strain, suggests that the causal organism is a new species within the genus Monilinia that we propose be classified as Monilinia rubi. A transition from designation as a basidiomycete fungus to an ascomycete fungus could have implications on chemical management decisions, as well as the assumptions made about cell structure and the pathogen's putative life cycle.

A Novel Signaling Pathway Connects Thiamine Biosynthesis, Bacterial Respiration, and Production of the Exopolysaccharide Amylovoran in Erwinia amylovora.

Erwinia amylovora is a plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. This bacterium colonizes host vascular tissues via the production of exopolysaccharides (EPSs) including amylovoran. It is well-established that the nearly ubiquitous plasmid pEA29 of E. amylovora is an essential virulence factor, but the underlying mechanism remains uncharacterized. Here, we demonstrated that pEA29 was required for E. amylovora to produce amylovoran and to form a biofilm, and this regulation was dependent on the thiamine biosynthesis operon thiOSGF. We then conducted carbohydrate and genetic analyses demonstrating that the thiamine-mediated effect on amylovoran production was indirect, as cells lacking thiOSGF produced an EPS that did not contain glucuronic acid, one of the key components of amylovoran, whereas the transcriptional activity and RNA levels of the amylovoran biosynthesis genes were not altered. Alternatively, addition of exogenous thiamine restored amylovoran production in the pEA29-cured strain of E. amylovora and positively impacted amylovoran production in a dose-dependent manner. Individual deletion of several chromosomal thiamine biosynthesis genes also affected amylovoran production, implying that a complete thiamine biosynthesis pathway is required for the thiamine-mediated effect on amylovoran production in E. amylovora. Finally, we determined that an imbalanced tricarboxylic acid cycle negatively affected amylovoran production, which was restored by addition of exogenous thiamine or overexpression of the thiOSGF operon. In summary, our report revealed a novel signaling pathway that impacts E. amylovora virulence in which thiamine biosynthesis enhances bacterial respiration that provides energetic requirements for the biosynthesis of EPS amylovoran.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Characterization of Streptomycin Resistance in Isolates of Erwinia amylovora in California.

In surveys from 2006 to 2014, streptomycin resistance in Erwinia amylovora from pear-growing areas in California declined from very high incidence in 2006 and 2007 to very low incidence in 2013 and 2014. The majority of resistant strains were designated as moderately resistant-low (MR-L), and were almost exclusively found in Sacramento County, whereas highly resistant (HR) strains were only recovered in Sutter-Yuba and San Joaquin counties. Resistance of HR strains was associated with a mutation in codon 43 of the chromosomal rpsL gene that results in a change from lysine to arginine, the same mutation that was originally reported for resistant strains from California in the mid-1970s. MR-L strains were found to harbor the strA-strB streptomycin resistance genes on transposon Tn5393a. This transposon lacks insertion sequence IS1133 that provides a promoter for efficient expression of strA-strB, resulting in lower minimum inhibitory concentrations of MR-L strains compared with those from other locations that harbor strA-strB on Tn5393::IS1133. In contrast to previously described plasmid-mediated resistance where Tn5393 is inserted in pEa34, or pEA29, Tn5393a in MR-L strains was located on plasmid pEU30. This plasmid was first described in E. amylovora from the western United States but was not associated with streptomycin resistance determinants previously. We hypothesize that Tn5393a was introduced into an E. amylovora strain carrying pEU30 and transposed into that plasmid. This hypothesis was supported by clustered regularly interspaced short palindromic repeat (CRISPR) sequence analysis that showed that two MR-L strains share the same CRISPR1 pattern as a streptomycin-sensitive strain. With current low resistance levels in California growing regions, streptomycin could be successfully used again, but applications per season should be limited and the antibiotic should be mixed and rotated with different modes of action.

Evaluation of kasugamycin for fire blight management, effect on nontarget bacteria, and assessment of kasugamycin resistance potential in Erwinia amylovora.

The emergence and spread of streptomycin-resistant strains of Erwinia amylovora in Michigan has necessitated the evaluation of new compounds effective for fire blight control. The aminoglycoside antibiotic kasugamycin (Ks) targets the bacterial ribosome and is particularly active against E. amylovora. The efficacy of Ks formulated as Kasumin 2L for control of fire blight was evaluated in six experiments conducted over four field seasons in our experimental orchards in East Lansing, MI. Blossom blight control was statistically equivalent to the industry standard streptomycin in all experiments. E. amylovora populations remained constant on apple flower stigmas pretreated with Kasumin and were ≈100-fold lower than on stigmas treated with water. Kasumin applied to apple trees in the field also resulted in a 100-fold reduced total culturable bacterial population compared with trees treated with water. We performed a prospective analysis of the potential for kasugamycin resistance (Ks(R)) development in E. amylovora which focused on spontaneous resistance development and acquisition of a transferrable Ks(R) gene. In replicated lab experiments, the development of spontaneous resistance in E. amylovora to Ks at 250 or 500 ppm was not observed when cells were directly plated on medium containing high concentrations of the antibiotic. However, exposure to increasing concentrations of Ks in media (initial concentration 25 µg ml(-1)) resulted in the selection of Ks resistance (at 150 µg ml(-1)) in the E. amylovora strains Ea110, Ea273, and Ea1189. Analysis of mutants indicated that they harbored mutations in the kasugamycin target ksgA gene and that all mutants were impacted in relative fitness observable through a reduced growth rate in vitro and decreased virulence in immature pear fruit. The possible occurrence of a reservoir of Ks(R) genes in orchard environments was also examined. Culturable gram-negative bacteria were surveyed from six experimental apple orchards that had received at least one Kasumin application. In total, 401 Ks(R) isolates (42 different species) were recovered from apple flowers and leaves and orchard soil samples. Although we have not established the presence of a transferrable Ks(R) gene in orchard bacteria, the frequency, number of species, and presence of Ks(R) enterobacterial species in orchard samples suggests the possible role of nontarget bacteria in the future transfer of a Ks(R) gene to E. amylovora. Our data confirm the importance of kasugamycin as an alternate antibiotic for fire blight management and lay the groundwork for the development and incorporation of resistance management strategies.

Genetic analysis of streptomycin-resistant (Sm(R)) strains of Erwinia amylovora suggests that dissemination of two genotypes is responsible for the current distribution of Sm(R) E. amylovora in Michigan.

Streptomycin-resistant (Sm(R)) strains of the fire blight pathogen Erwinia amylovora were first isolated in southwest Michigan in 1991. Since that time, resistant strains have progressed northward to other apple-producing regions in the state. A total of 98.7% of Sm(R) strains isolated between 2003 and 2009 in Michigan harbored the strA-strB genes on transposon Tn5393. strA and strB encode phosphotransferase enzymes that modify streptomycin to a nonbactericidal form. Mutational resistance to streptomycin, caused by a point mutation-mediated target-site alteration of the ribosomal S12 protein, occurred in 1.3% of E. amylovora strains from Michigan. Tn5393 was originally introduced to E. amylovora on the plasmid pEa34; thus, the first Sm(R) strains isolated contained both pEa34 and the ubiquitous nonconjugative plasmid pEA29. More recently, we have observed Sm(R) strains in which Tn5393 is present on pEA29, suggesting that the transposon has moved via transposition from pEa34 to pEA29. Almost all of the strains containing Tn5393 on pEA29 had lost pEa34. Of 210 pEA29::Tn5393 plasmids examined, the transposon was inserted at either nucleotide position 1,515 or 17,527. Both of these positions were in noncoding regions of pEA29. Comparative sequencing of the housekeeping genes groEL and potentially variable sequences on pEA29 was done in an attempt to genetically distinguish Sm(R) strains from streptomycin-sensitive (Sm(S)) strains isolated in Michigan. Only 1 nucleotide difference within the total 2,660 bp sequenced from each strain was observed in 2 of 29 strains; multiple sequence differences were observed between the Michigan strains and E. amylovora control strains isolated in the western United States or from Rubus spp. Alterations in virulence observable using an immature pear fruit assay were detected in three of eight Sm(R) strains examined. Our current genetic data indicate that only two Sm(R) strain genotypes (strains containing pEA29::Tn5393 with Tn5393 inserted at either nucleotide position 1,515 or 17,527 on the plasmid) are responsible for the dissemination of Tn5393-encoded streptomycin resistance in Michigan, and that the Sm(R) and Sm(S) strains in Michigan compose a homogenous group.

Control of fire blight (Erwinia amylovora) on apple trees with trunk-injected plant resistance inducers and antibiotics and assessment of induction of pathogenesis-related protein genes.

Management of fire blight is complicated by limitations on use of antibiotics in agriculture, antibiotic resistance development, and limited efficacy of alternative control agents. Even though successful in control, preventive antibiotic sprays also affect non-target bacteria, aiding the selection for resistance which could ultimately be transferred to the pathogen Erwinia amylovora. Trunk injection is a target-precise pesticide delivery method that utilizes tree xylem to distribute injected compounds. Trunk injection could decrease antibiotic usage in the open environment and increase the effectiveness of compounds in fire blight control. In field experiments, after 1-2 apple tree injections of either streptomycin, potassium phosphites (PH), or acibenzolar-S-methyl (ASM), significant reduction of blossom and shoot blight symptoms was observed compared to water injected control trees. Overall disease suppression with streptomycin was lower than typically observed following spray applications to flowers. Trunk injection of oxytetracycline resulted in excellent control of shoot blight severity, suggesting that injection is a superior delivery method for this antibiotic. Injection of both ASM and PH resulted in the significant induction of PR-1, PR-2, and PR-8 protein genes in apple leaves indicating induction of systemic acquired resistance (SAR) under field conditions. The time separating SAR induction and fire blight symptom suppression indicated that various defensive compounds within the SAR response were synthesized and accumulated in the canopy. ASM and PH suppressed fire blight even after cessation of induced gene expression. With the development of injectable formulations and optimization of doses and injection schedules, the injection of protective compounds could serve as an effective option for fire blight control.

Genetic Analysis of a Pathogenic Erwinia sp. Isolated from Pear in Japan.

ABSTRACT Four Erwinia strains, originally isolated in Japan from pear trees with bacterial shoot blight symptoms, were analyzed to determine their genetic relationship with Erwinia amylovora and E. pyrifoliae. When genomes were characterized with amplified fragment length polymorphism markers and by comparative groEL sequence analysis, the Japanese Erwinia sp. and South Korean E. pyrifoliae strains were placed in the same group, which was phylogenetically distinct from a group of 15 strains of E. amylovora. Sequencing of the 29,593-bp plasmid pEJ30 from Erwinia strain Ejp556 revealed that this plasmid was nearly identical to plasmid pEP36 from E. pyrifoliae and was closely related to the nontransferable ubiquitous plasmid pEA29 from E. amylovora. Twenty-one presumptive genes and their order in pEP36 were highly conserved in pEJ30; however, transposon Tn5394, which was present in pEP36, was not found in pEJ30. Short-sequence DNA repeats were conserved between pEJ30 and pEP36, and were different from short-sequence repeats in pEA29. Despite base-pair mismatches, primer pairs used in pEA29 polymerase chain reaction assays for E. amylovora amplified plasmid DNA from the Japanese Erwinia Ejp556 and Ejp562. Like E. pyrifoliae and a few strains of E. amylovora, Japanese Erwinia Ejp617 contained plasmids related to E. pyrifoliae ColE1-related plasmid pEP2.6. Based on these genetic analyses, we conclude that the Erwinia pathogen of pear in Japan is closely related to E. pyrifoliae and that both of these pathogens are demonstrably distinct from E. amylovora.

Erwinia amylovora CRISPR elements provide new tools for evaluating strain diversity and for microbial source tracking.

Clustered regularly interspaced short palindromic repeats (CRISPRs) comprise a family of short DNA repeat sequences that are separated by non repetitive spacer sequences and, in combination with a suite of Cas proteins, are thought to function as an adaptive immune system against invading DNA. The number of CRISPR arrays in a bacterial chromosome is variable, and the content of each array can differ in both repeat number and in the presence or absence of specific spacers. We utilized a comparative sequence analysis of CRISPR arrays of the plant pathogen Erwinia amylovora to uncover previously unknown genetic diversity in this species. A total of 85 E. amylovora strains varying in geographic isolation (North America, Europe, New Zealand, and the Middle East), host range, plasmid content, and streptomycin sensitivity/resistance were evaluated for CRISPR array number and spacer variability. From these strains, 588 unique spacers were identified in the three CRISPR arrays present in E. amylovora, and these arrays could be categorized into 20, 17, and 2 patterns types, respectively. Analysis of the relatedness of spacer content differentiated most apple and pear strains isolated in the eastern U.S. from western U.S. strains. In addition, we identified North American strains that shared CRISPR genotypes with strains isolated on other continents. E. amylovora strains from Rubus and Indian hawthorn contained mostly unique spacers compared to apple and pear strains, while strains from loquat shared 79% of spacers with apple and pear strains. Approximately 23% of the spacers matched known sequences, with 16% targeting plasmids and 5% targeting bacteriophage. The plasmid pEU30, isolated in E. amylovora strains from the western U.S., was targeted by 55 spacers. Lastly, we used spacer patterns and content to determine that streptomycin-resistant strains of E. amylovora from Michigan were low in diversity and matched corresponding streptomycin-sensitive strains from the background population.

Effect of a waaL mutation on lipopolysaccharide composition, oxidative stress survival, and virulence in Erwinia amylovora.

Erwinia amylovora, the causal agent of fire blight, is an enterobacterial pathogen of Rosaceous plants including apple and pear. We have been studying the response of E. amylovora to oxidative stress because, during infection, the bacterium elicits an oxidative burst response in host plants. During the screening of a transposon mutant library for hydrogen peroxide sensitivity, we identified a mutant carrying an insertion in waaL, a gene involved in lipopolysaccharide biosynthesis, that was more sensitive to hydrogen peroxide than the parental wild-type strain. We also confirmed that a waaL mutant of Pseudomonas aeruginosa exhibited an increased sensitivity to hydrogen peroxide compared with the wild-type strain. The E. amylovora waaL mutant was also reduced in virulence, showed a decrease in twitching motility, and was more sensitive to polymyxin B than the wild type. Each of these phenotypes was complemented by the cloned waaL gene. Our results highlight the importance of the lipopolysaccharide layer to virulence in E. amylovora and the unexpected finding of an additional function of lipopolysaccharide in protection from oxidative stress in E. amylovora and P. aeruginosa.

Nucleotide sequences, genetic organization, and distribution of pEU30 and pEL60 from Erwinia amylovora.

The nucleotide sequences, genetic organization, and distribution of plasmids pEU30 (30,314 bp) and pEL60 (60,145 bp) from the plant pathogen Erwinia amylovora are described. The newly characterized pEU30 and pEL60 plasmids inhabited strains isolated in the western United States and Lebanon, respectively. The gene content of pEU30 resembled plasmids found in plant-associated bacteria, while that of pEL60 was most similar to IncL/M plasmids inhabiting enteric bacteria.

Relatedness of chromosomal and plasmid DNAs of Erwinia pyrifoliae and Erwinia amylovora.

The plant pathogen Erwinia pyrifoliae has been classified as a separate species from Erwinia amylovora based in part on differences in molecular properties. In this study, these and other molecular properties were examined for E. pyrifoliae and for additional strains of E. amylovora, including strains from brambles (Rubus spp.). The nucleotide composition of the internal transcribed spacer (ITS) region was determined for six of the seven 16S-23S rRNA operons detected in these species with a 16S rRNA gene probe. Each species contained four operons with a tRNA(Glu) gene and two with tRNA(Ile) and tRNA(Ala) genes, and analysis of the operons from five strains of E. amylovora indicated a high degree of ITS variability among them. One tRNA(Glu)-containing operon from E. pyrifoliae Ep1/96 was identical to one in E. amylovora Ea110, but three tRNA(Glu) operons and two tRNA(Ile) and tRNA(Ala) operons from E. pyrifoliae contained unique nucleotide changes. When groEL sequences were used for species-specific identification, E. pyrifoliae and E. amylovora were the closest phylogenetic relatives among a set of 12 bacterial species. The placement of E. pyrifoliae distinct from E. amylovora corroborated molecular hybridization data indicating low DNA-DNA similarity between them. Determination of the nucleotide sequence of plasmid pEP36 from E. pyrifoliae Ep1/96 revealed a number of presumptive genes that matched genes previously found in pEA29 from E. amylovora and similar organization for the genes and origins of replication. Also, pEP36 and pEA29 were incompatible with clones containing the reciprocal origin regions. Finally, the ColE1-like plasmid pEP2.6 from strain Ep1/96 contained sequences found in small plasmids in E. amylovora strains IL-5 and IH3-1.