Apple growers in the Mid-Atlantic region of the United States have been reporting an increase in losses to bitter rot of apple and are requesting up-to-date management recommendations. Management is ...complicated by variations in apple cultivar susceptibility, temperature, rainfall, and biology of the Colletotrichum spp. that cause bitter rot. Over 500 apple fruit with bitter rot were obtained from 38 orchards across the Mid-Atlantic and the causal species were identified as Colletotrichum fioriniae and C. nymphaeae of the C. acutatum species complex and C. chrysophilum, C. noveboracense, C. siamense, C. fructicola, C. henanense, and C. gloeosporioides sensu stricto of the C. gloeosporioides species complex, the latter two being first reports. Species with faster in vitro growth rates at higher temperatures were more abundant in warmer regions of the Mid-Atlantic, while those with slower growth rates at higher temperatures were more abundant in cooler regions. Regional bloom dates are earlier and weather data show a gradual warming trend that likely influenced but was not necessarily the main cause of the recent increase in bitter rot in the region. A grower survey of apple cultivar susceptibility showed high variation, with the increase in acres planted to the highly susceptible cultivar Honeycrisp broadly corresponding to the increase in reports of bitter rot. These results form a basis for future studies on the biology and ecology of the Colletotrichum spp. responsible, and suggest that integrated bitter rot management must begin with selection of less-susceptible apple cultivars.
Apple bitter rot caused by Colletotrichum species is a growing problem worldwide. Colletotrichum spp. are economically important but taxonomically un-resolved. Identification of Colletotrichum spp. ...is critical due to potential species-level differences in pathogenicity-related characteristics. A 400-isolate collection from New York apple orchards were morphologically assorted to two groups, C. acutatum species complex (CASC) and C. gloeosporioides species complex (CGSC). A sub-sample of 44 representative isolates, spanning the geographical distribution and apple varieties, were assigned to species based on multi-locus phylogenetic analyses of nrITS, GAPDH and TUB2 for CASC, and ITS, GAPDH, CAL, ACT, TUB2, APN2, ApMat and GS genes for CGSC. The dominant species was C. fioriniae, followed by C. chrysophilum and a novel species, C. noveboracense, described in this study. This study represents the first report of C. chrysophilum and C. noveboracense as pathogens of apple. We assessed the enzyme activity and fungicide sensitivity for isolates identified in New York. All isolates showed amylolytic, cellulolytic and lipolytic, but not proteolytic activity. C. chrysophilum showed the highest cellulase and the lowest lipase activity, while C. noveboracense had the highest amylase activity. Fungicide assays showed that C. fioriniae was sensitive to benzovindiflupyr and thiabendazole, while C. chrysophilum and C. noveboracense were sensitive to fludioxonil, pyraclostrobin and difenoconazole. All species were pathogenic on apple fruit with varying lesion sizes. Our findings of differing pathogenicity-related characteristics among the three species demonstrate the importance of accurate species identification for any downstream investigations of Colletotrichum spp. in major apple growing regions.
Fire blight is a devastating disease of apple and pear caused by the bacterium Erwinia amylovora. One of its main symptoms is canker formation on perennial tissues which may lead to the death of ...limbs and/or the entire tree. E. amylovora overwinters in cankers which play an important role in initiating fire blight epidemics. However, knowledge of pathogen biology in cankers is scarce, in part due to limitations of classical microbiology methods and the inability of most molecular techniques to distinguish live from dead cells. In this work, a viability digital PCR (v-dPCR) protocol using propidium monoazide (PMA) was developed, allowing for the first time the selective detection and absolute quantification of E. amylovora live cells in apple and pear cankers collected in two time periods. Some key factors affecting the v-dPCR performance were the maceration buffer composition, the target DNA amplicon length, the thermal cycle number and the use of sodium dodecyl sulfate or PMA enhancer for Gram-negative bacteria to improve the effect of PMA. In the future, this methodology could shed light on E. amylovora population dynamics in cankers and provide clues on the effect of management practices, host cultivar, host water/nutritional status, etc., on bacterial survival.
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.
Erwinia amylovora
causes fire blight, a disease responsible for enormous economic losses in the pome fruit-producing areas where it is present. Despite the abundant research on fire blight, ...information about
E. amylovora
population dynamics and survival in fire blight cankers and the plant defense responses to this pathogen in the infected bark are limited. In our study, we obtained fire blight cankers in apple, pear, and Asian pear cultivars showing differing resistance to the disease by shoot inoculation with
E. amylovora
. We collected cankers from irrigated and non-irrigated trees every 3 months in two independent field experiments and analyzed samples by viability digital PCR. We also assessed the expression of pathogenicity-related (
PR
) genes in the bark of selected apple and Asian pear cultivars. A logistic regression analysis revealed the impact of environmental and host factors on
E. amylovora
detection rates in cankers. The chances of detecting live
E. amylovora
cells in cankers increased significantly in those collected from irrigated trees, in July, and/or during an experiment performed in a year with an expected average rainfall when compared to samples from non-irrigated trees, collected in January, and/or during an experiment performed under environmental conditions dominated by drought. We found a positive correlation between the pathogen detection rates in cankers and the host resistance to fire blight that might be explained by lower
E. amylovora
survival rates in more damaged tissues of susceptible hosts. The genes
PR-1, PR-2, PR-5,
and
PR-8
were induced in the bark surrounding apple and Asian pear fire blight cankers. Our study, involving the analysis of more than 800 canker samples, provides new knowledge about the fire blight disease cycle and lays the foundation for improved fire blight management and eradication strategies in pome fruit orchards.
Apple growers in the Mid-Atlantic region of the U.S.A. have reported increased losses to bitter rot of apple. We tested the hypothesis that this increase is because the
population has developed ...resistance to commonly used single-mode-of-action (single-MoA) fungicides. We screened 220
isolates obtained from 38 apple orchards in the Mid-Atlantic region for resistance to 11 fungicides in Fungicide Resistance Action Committee (FRAC) groups 1, 7, 9, 11, 12, and 29. Eleven (5%) of these isolates were resistant to FRAC group 1 with confirmed β-tubulin E198A mutations, and two (<1%) were also resistant to FRAC group 11 with confirmed cytochrome-
G143A mutations. Such low frequencies of resistant isolates indicate that fungicide resistance is unlikely to be the cause of any regional increase in bitter rot. A subsample of isolates was subsequently tested in vitro for sensitivity to every single-MoA fungicide registered for apple in the Mid-Atlantic U.S.A. (22 fungicides; FRAC groups 1, 3, 7, 9, 11, 12, and 29), and 13 fungicides were tested in field trials. These fungicides varied widely in efficacy both within and between FRAC groups. Comparisons of results from our in vitro tests with results from our field trials and other field trials conducted across the eastern U.S.A. suggested that EC
values (concentrations that reduce growth by 25%) are better predictors of fungicide efficacy in normal field conditions than EC
values. We present these results as a guideline for choosing single-MoA fungicides for bitter rot control in the Mid-Atlantic U.S.A.
Bitter rot, caused by
species, is one of the most devastating summer rot diseases affecting apple production in the Eastern United States. Given the differences in virulence and fungicide sensitivity ...levels between organisms belonging to the
species complex (CASC) and the
species complex (CGSC), monitoring their diversity, geographic distribution, and frequency % are essential for successful bitter rot management. In a 662-isolate collection from apple orchards in Virginia, isolates from CGSC were dominant (65.5%) in comparison to the CASC (34.5%). In a sub-sample of 82 representative isolates, using morphological and multi-locus phylogenetic analyses, we identified
(26.2%),
(15.6%),
(0.8%) and
(0.8%) from CGSC and
(22.1%) and
(1.6%) from CASC. The dominant species were
, followed by
and
.
followed by
developed the largest and deepest rot lesions on 'Honeycrisp' fruit in our virulence tests. Detached fruit of 9 apple cultivars and one wild accession (
) were harvested early and late season and tested in controlled conditions for their susceptibility to
and
. All cultivars were susceptible to both representative bitter rot species, with 'Honeycrisp' fruit being the most susceptible and
, accession PI 369855, being the most resistant. We demonstrate that the frequency and prevalence of species in
complexes are highly variable in the Mid-Atlantic and provide regional-specific data on apple cultivar susceptibility. Our findings are necessary for the successful management of bitter rot as an emerging and persistent problem in apple production both pre- and postharvest.
Bitter rot of apple is an economically important worldwide disease caused by different
species, depending on many factors such as climate, geography, other hosts, and crop management practices. ...Culture, morphology, and single-locus sequencing-based methods for identifying the
species are severely limited in effectiveness, while the multilocus sequence typing methods available for delineating species are costly, time-intensive, and require high expertise. We developed species-specific hydrolysis probe real-time PCR assays for the following nine
species causing bitter rot in the Mid-Atlantic U.S.A.:
,
,
,
s.s.,
,
and
from the
species complex, and
and
from the
species complex. After searching 14 gene regions, we designed primers and probes in 5 of them for the nine target species. Four primer-probe set pairs were able to be duplexed. Sensitivity tests showed as little as 0.5 pg DNA were detectable. These real-time PCR assays will provide rapid and reliable identification of these key
species and will be critically important for studies aiming to elucidate their biology, epidemiology, and management on apples as the number one produced and consumed tree fruit in the U.S.A.
The bacterium
causes fire blight and continues to threaten global commercial apple and pear production. Conventional microbiology techniques cannot accurately determine the presence of live pathogen ...cells in fire blight cankers. Several factors may prevent
from growing on solid culture media, including competing microbiota and the release of bacterial-growth-inhibitory compounds by plant material during sample processing. We previously developed a canker processing methodology and a chip-based viability digital PCR (v-dPCR) assay using propidium monoazide (PMA) to bypass these obstacles. However, sample analysis was still time-consuming and physically demanding. In this work, we improved the previous protocol using an automatic tissue homogenizer and transferred the chip-based v-dPCR to the BioRad QX200 droplet dPCR (ddPCR) platform. The improved sample processing method allowed the simultaneous, fast, and effortless processing of up to six samples. Moreover, the transferred v-ddPCR protocol was compatible with the same PMA treatment and showed a similar dynamic range, from 7.2 × 10
to 7.6 × 10
cells mL
, as the previous v-dPCR. Finally, the improved protocol allowed, for the first time, the detection of
viable but nonculturable (VBNC) cells in cankers and bark tissues surrounding cankers. Our v-ddPCR assay will enable new ways to evaluate resistant pome fruit tree germplasm, further dissect the
life cycle, and elucidate
physiology, epidemiology, and new options for canker management.
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