Fungicide Resistance of Alternaria alternata and A. arborescens Isolates from Mandarin Fruit and Its Influence on Control of Postharvest Alternaria Rot.

Alternaria rot, caused by Alternaria alternata and A. arborescens, is one of the common postharvest diseases affecting mandarin fruit in California. Fungicide resistance profilings of A. alternata and A. arborescens to quinone outside inhibitors (QoIs), fludioxonil, pyrimethanil, imazalil, and propiconazole were examined in this study. Of the 100 isolates of A. alternata and A. arborescens, 40 were identified as resistant to QoI fungicides according to a PCR-restriction fragment length polymorphism based on a cytochrome b partial gene. Effective concentrations of fludioxonil that caused a 50% reduction in fungal growth relative to the control (EC50) were 0.089 ± 0.020 and 0.101 ± 0.032 µg/ml for 43 A. alternata and 19 A. arborescens isolates, respectively. EC50 values of pyrimethanil, imazalil, and propiconazole for the 70 A. alternata isolates tested were 0.373 ± 0.161, 0.492 ± 0.133, and 1.135 ± 0.407 µg/ml, respectively. EC50 values of pyrimethanil, imazalil, and propiconazole for 30 A. arborescens isolates were 0.428 ± 0.190, 0.327 ± 0.180, and 0.669 ± 0.452 µg/ml, respectively. Control tests on mandarin fruit inoculated with representative isolates of both species showed that fludioxonil, pyrimethanil, imazalil and propiconazole significantly reduced disease incidence and severity. Azoxystrobin significantly reduced Alternaria rot severity and incidence on mandarin fruit inoculated with sensitive isolates but not with resistant isolates, regardless of Alternaria spp. There were no significant differences in Alternaria rot control effectiveness if treatment with any of the fungicides tested was delayed after inoculation by 6 or 12 h. These results could help in the development of postharvest fungicide programs to control Alternaria rot on mandarin fruit during storage.

Fungicide Resistance and Host Influence on Population Structure in Botrytis spp. from Specialty Crops in California.

Botrytis is an important genus of plant pathogens causing pre- and postharvest disease on diverse crops worldwide. This study evaluated Botrytis isolates collected from strawberry, blueberry, and table grape berries in California. Isolates were evaluated for resistance to eight different fungicides, and 60 amplicon markers were sequenced (neutral, species identification, and fungicide resistance associated) distributed across 15 of the 18 B. cinerea chromosomes. Fungicide resistance was common among the populations, with resistance to pyraclostrobin and boscalid being most frequent. Isolates from blueberry had resistance to the least number of fungicides, whereas isolates from strawberry had resistance to the highest number. Host and fungicide resistance-specific population structure explained 12 and 7 to 26%, respectively, of the population variability observed. Fungicide resistance was the major driver for population structure, with select fungicides explaining up to 26% and multiple fungicide resistance explaining 17% of the variability observed. Shared and unique significant single-nucleotide polymorphisms (SNPs) associated with host and fungicide (fluopyram, thiabendazole, pyraclostrobin, and fenhexamid) resistance-associated population structures were identified. Although overlap between host and fungicide resistance SNPs were detected, unique SNPs suggest that both host and fungicide resistance play an important role in Botrytis population structure.

Fungicide Resistance in Alternaria alternata from Blueberry in California and Its Impact on Control of Alternaria Rot.

Alternaria rot caused by Alternaria alternata is one of the major postharvest diseases affecting blueberries in California. The sensitivity profiles of A. alternata from blueberry field to quinone outside inhibitors (QoIs), boscalid, fluopyram, fludioxonil, cyprodinil, and polyoxin D in California were examined in this study. EC50 values of 51 A. alternata isolates for boscalid varied greatly among the isolates, ranging from 0.265 to >100 µg/ml. EC50 values of 51 A. alternata isolates to fluopyram, fludioxonil, cyprodinil, and polyoxin D were 5.188 ± 7.118, 0.078 ± 0.021, 0.465 ± 0.302, and 6.238 ± 7.352 µg/ml, respectively. In total, 143 isolates were screened for resistance at 5 and 10 µg/ml for fludioxonil, cyprodinil, and fluopyram, 10 µg/ml for polyoxin D, and 10 and 50 µg/ml for boscalid. Based on the published discriminatory concentrations for phenotyping resistance, of the 143 isolates, all were considered resistant to boscalid; 32, 69, and 42 were sensitive, low resistant, and resistant to fluopyram, respectively; and all were sensitive to fludioxonil and cyprodinil. In a PCR-restriction fragment length polymorphism method for phenotyping, 60 out of the 143 isolates were classified as resistant to QoIs. Control tests on detached blueberry fruit inoculated with different Alternaria isolates showed that fludioxonil and cyprodinil significantly reduced disease incidence and severity; however, pyraclostrobin, boscalid, fluopyram, and polyoxin D significantly reduced only disease severity. The obtained results will be helpful in making decisions on fungicide programs to control A. alternata isolates with resistance or reduced sensitivities to multiple fungicides.

Baseline Sensitivity of Alternaria alternata and A. arborescens to Natamycin and Control of Alternaria Rot on Stored Mandarin Fruit.

Alternaria rot caused by Alternaria alternata and A. arborescens is one of the major postharvest diseases on mandarin fruit in California. In this study, natamycin, a newly registered biofungicide, was evaluated for its potential as a postharvest treatment to control Alternaria rot on mandarin fruit. The baseline sensitivities of A. alternata and A. arborescens to natamycin were determined. Effective concentration inhibiting 50% of fungal growth (EC50) values of natamycin for 70 A. alternata isolates ranged from 0.694 to 1.275 µg/ml (mean = 0.921 µg/ml) in a conidial germination assay and from 2.001 to 3.788 µg/ml (mean = 2.797 µg/ml) for 40 A. alternata isolates in a mycelial growth assay. EC50 values of natamycin for 30 A. arborescens isolates ranged from 0.698 to 1.203 µg/ml (mean = 0.923 µg/ml) in a conidial germination assay and from 2.035 to 3.368 µg/ml (mean = 2.658 µg/ml) for 20 A. arborescens isolates in a mycelial growth assay. Control tests on detached mandarin fruit showed that natamycin at both low (460 µg/ml) and high (920 µg/ml) recommended rates significantly reduced disease incidence and severity on mandarin fruit inoculated with Alternaria isolates, regardless of species. High rate of natamycin significantly reduced disease incidence and severity compared with the nontreated control even when natamycin treatment was delayed for 6, 12, and 18 h after inoculation. Our results suggested that natamycin can be an effective postharvest fungicide for control of Alternaria rot on mandarin fruit.

Effects of Peroxyacetic Acid on Postharvest Diseases and Quality of Blueberries.

Postharvest diseases are a limiting factor in the storage of fresh blueberries. Gray mold caused by Botrytis cinerea and Alternaria rot caused by Alternaria spp. are important postharvest diseases in blueberries grown in California. Control of these fungal pathogens is generally dependent on preharvest sprays of synthetic fungicides, but in California multiple fungicide resistance has already developed in those pathogens, leading to the failure of disease control. Therefore, alternatives to synthetic fungicides are needed for the control of postharvest diseases. Peroxyacetic acid (PAA) is a disinfectant agent that poses low risk to human health. In this study, we evaluated the effects of postharvest use of PAA at 24 µl liter-1 and 85 µl liter-1 on fruit decay caused by fungal pathogens and quality of stored blueberry fruit. PAA treatment was applied to four cultivars over three seasons using two methods, dipping or spraying. Dipping blueberries compared with spraying them with PAA and its application at 85 µl liter-1 were the most effective treatments. For example, when applied to 'Snowchaser' blueberries, this combination reduced naturally occurring decay after 4 weeks of storage at 0 to 1°C from 14.3% among water-treated controls to 2.7% in 2018, and from 25.7% among water-treated controls to 8.6% in 2020. In general, PAA did not adversely affect fruit quality or sensory quality of blueberries. Postharvest use of PAA appears to be a promising means to reduce postharvest decay of blueberries. To reliably obtain an acceptable level of disease control, the best use of PAA may be in combination with other practices rather than using it alone.

Phylogenetic, Morphological, and Pathogenic Characterization of Alternaria Species Associated With Fruit Rot of Mandarin in California.

Alternaria rot caused by Alternaria species is one of the major postharvest diseases of mandarin fruit in California. The aims of this study were to identify these Alternaria species via phylogenetic analyses and morphological characteristics and test their pathogenicity on mandarin. Decayed mandarin fruits exhibiting Alternaria rot symptoms were collected from three citrus fruit packinghouses in the Central Valley of California. In total, 177 Alternaria isolates were obtained from decayed fruit and preliminarily separated into three groups representing three species (A. alternata, A. tenuissima, and A. arborescens) based on the colony characterization and sporulation patterns. To further identify these isolates, phylogenetic analysis was conducted based on DNA sequences of the second largest subunit of RNA polymerase II (RPB2), plasma membrane ATPase (ATPase), and Calmodulin gene regions in combination with morphological characters. Of the 177 isolates, 124 (70.1%) were identified as A. alternata, and 53 (29.9%) were A. arborescens. The isolates initially identified as A. tenuissima based on the morphological characteristics could not be separated from those of A. alternata in phylogenetic analysis and thus considered A. alternata. Pathogenicity tests showed that both Alternaria species were pathogenic on mandarin fruit at both 5°C and 20°C. Our results indicated that two Alternaria species, A. alternata and A. arborescens, were responsible for Alternaria rot of mandarin fruit in California, with A. arborescens causing fruit rot on mandarin being reported for the first time.

Botrytis spp.: A Contemporary Perspective and Synthesis of Recent Scientific Developments of a Widespread Genus that Threatens Global Food Security.

This perspective presents a synopsis of the topics contained in the Phytopathology Pathogen Spotlight on Botrytis spp. causing gray mold, including pathogen biology and systematics, genomic characterization of new species, perspectives on genome editing, and fungicide resistance. A timely breakthrough to engineer host plant resistance against the gray mold fungus has been demonstrated in planta and may augment chemical controls in the near future. While B. cinerea has garnered much of the research attention, other economically important Botrytis spp. have been identified and characterized via morphological and genome-based approaches. Gray mold control is achieved primarily through fungicide applications but resistance to various chemical classes is a major concern that threatens global plant health and food security. In this issue, new information on molecular mechanism(s) of fungicide resistance and ways to manage control failures are presented. Finally, a significant leap in fundamental pathogen biology has been achieved via development of CRISPR/Cas9 to assess gene function in the fungus which likely will spawn new control mechanisms and facilitate gene discovery studies.

Population Genetics and Fungicide Resistance of Botrytis cinerea on Vitis and Prunus spp. in California.

Botrytis cinerea, the causal agent of gray mold, has high genetic diversity and a broad host range. In Vitis sp. and Prunus spp., B. cinerea causes pre- and postharvest diseases, and fungicides are routinely applied to prevent yield loss. In total, 535 isolates of B. cinerea collected from Vitis sp. and Prunus spp. in 2012, 2016, and 2017 were genotyped using 18 microsatellite markers and the transposable elements (TEs) Boty and Flipper. Only nine of the polymorphic markers and the two TEs were considered informative and retained for the final analyses. Of the 532 isolates, 297 were tested for resistance to seven fungicides representing six Fungicide Resistance Action Committee classes. After clone correction, 295 multilocus genotype groups were retained across the 3 years in 326 individuals, and four genetic subpopulations were detected. High levels of clonality were observed across the dataset. Significant pairwise differentiation was detected among years, locations, and TE composition. However, most of the diversity observed was within a subpopulation and not among subpopulations. No genetic differentiation was detected among resistant and sensitive isolates for individual fungicide classes. When resistance to the total number of fungicides was compared, regardless of the fungicide class, significant differentiation was detected among isolates that are resistant to two fungicide classes and those resistant to three or four fungicide groups. Fungicide resistance frequencies were stable for most chemistries evaluated with the exception of fluopyram, which increased from 2012 to 2016/2017.

Timing of Perennial Canker Development in Apple Trees Caused by Neofabraea perennans and Neofabraea kienholzii.

Members of the genera Neofabraea and Phlyctema have been reported to incite canker diseases of apple trees and a postharvest decay of apple fruit referred to as "bull's-eye rot." Neofabraea kienholzii was recently identified as participating in the bull's-eye rot disease complex of apple and other pome fruit. In this study, apple twigs inoculated with N. kienholzii were shown to develop symptoms of a canker disease closely resembling perennial canker of pome fruit trees caused by N. perennans. Cankers resulting from infection by either Neofabraea spp. were more likely to be induced when twig inoculations occurred in October, and to a lesser degree in April, compared with all other inoculation dates evaluated in this study. Although N. kienholzii tended to induce cankers that were smaller in size compared with N. perennans, both pathogens shared similar seasonal trends in the initiation and expansion of tree cankers. N. perennans and N. kienholzii were recovered from inoculated twigs 6 months postinoculation regardless of when inoculations were conducted, indicating that both pathogens can survive on diseased twigs year-round. In addition, acervuli were observed more often on twigs inoculated in September and April compared with those inoculated in other months. Data from this work should help further our understanding of the epidemiology of N. kienholzii. This information also highlights the importance of proper branch pruning, canker removal, and aphid control. Such management activities should be conducted in a manner that helps minimize further spread of the pathogen.

Detection and Identification of Six Monilinia spp. Causing Brown Rot Using TaqMan Real-Time PCR from Pure Cultures and Infected Apple Fruit.

Brown rot is a severe disease affecting stone and pome fruit. This disease was recently confirmed to be caused by the following six closely related species: Monilinia fructicola, M. laxa, M. fructigena, Monilia polystroma, M. mumecola, and M. yunnanensis. Because of differences in geographic distributions, some of these species are important quarantine pathogens in certain countries. In this study, we developed TaqMan real-time polymerase chain reaction (PCR) assays to detect and identify the six species. Primer pairs and probes were designed for Monilinia fructicola, M. fructigena, M. laxa, and Monilia polystroma based on sequence differences in the laccase-2 genes. Additionally, based on sequence differences in the elongation factor genes, primer pairs and probes were designed for Monilia mumecola and M. yunnanensis. The real-time PCR assays were able to specifically identify the target pathogens, with detection limits of 10 to 100 fg of DNA, which is equivalent to one to seven conidia. The assays were also able to detect the target pathogens in a mixed DNA sample comprising all six Monilinia spp. and related species. The real-time PCR assays accurately detected target fungi from infected apple fruit. Furthermore, the identification results were consistent with those of traditional morphological methods.

Pi (Spleen)-deficiency syndrome in tumor microenvironment is the pivotal pathogenesis of colorectal cancer immune escape / 中国结合医学杂志

Cancer immunoediting consists of three sequential phases: elimination, equilibrium, and escape. For colorectal adenoma-carcinoma sequence, the adenoma dysplastic progression may represent an equilibrium phase and the cancer stage as escape phase. Immune system eliminates transformed enterocytes by destroying them at first, sculpts them at the same time and selects the variants subsequently that are no longer recognized and insensitive to immune effectors, and finally induces immunosuppressive state within the tumor microenvironment that facilitates immune escape and tumor outgrowth. Immunosuppression and inflammation are the two crucial features of Pi (Spleen)-deficiency. Classic quotations, immune evidence and clinical observations suggest that Spleen (but not other organs) deficiency is the key pathogenesis of colorectal cancer (CRC) microenvironment. Weakness of old age, immunosuppressive cytokines from chronic inflammation, tumor-derived immunosuppressive factors and surrendered immune cells-regulatory T cells, myeloid-derived suppressor cells and tumor associated macrophages (TAMs) constitutes CRC microenvironment of Pi-deficiency. Furthermore, excess in superficiality, such as phlegm stagnation, blood stasis and toxin accumulation are induced by chronic inflammation on the basis of asthenia in origin, an immunosuppressive state. Great masters of Chinese medicine emphasize that strengthen Pi is the chief therapeutic principle for CRC which receives good therapeutic effects. So, Pi-deficiency based syndrome is the pivotal pathogenesis of tumor microenvironment. The immunosuppressive microenvironment facilitates immune escape which play an important role in the transition from adenoma to adenocarcinoma. There are some signs that strengthen Pi based treatment has potential capacity to ameliorate tumor environment. It might be a novel starting point to explore the mechanism of strengthen Pi based therapy in the prevention and treatment of CRC through regulation of tumor environment and immunoediting.

Dynamics of verticillium species microsclerotia in field soils in response to fumigation, cropping patterns, and flooding.

Verticillium dahliae is a soilborne, economically significant fungal plant pathogen that persists in the soil for up to 14 years as melanized microsclerotia (ms). Similarly, V. longisporum is a very significant production constraint on members of the family Brassicaceae. Management of Verticillium wilt has relied on methods that reduce ms below crop-specific thresholds at which little or no disease develops. Methyl bromide, a broad-spectrum biocide, has been used as a preplant soil fumigant for over 50 years to reduce V. dahliae ms. However, reductions in the number of ms in the vertical and horizontal soil profiles and the rate at which soil recolonization occurs has not been studied. The dynamics of ms in soil before and after methyl bromide+chloropicrin fumigation were followed over 3 years in six 8-by-8-m sites in two fields. In separate fields, the dynamics of ms in the 60-cm-deep vertical soil profile pre- and postfumigation with methyl bromide+chloropicrin followed by various cropping patterns were studied over 4 years. Finally, ms densities were assessed in six 8-by-8-m sites in a separate field prior to and following a natural 6-week flood. Methyl bromide+chloripicrin significantly reduced but did not eliminate V. dahliae ms in either the vertical or horizontal soil profiles. In field studies, increases in ms were highly dependent upon the crop rotation pattern followed postfumigation. In the vertical soil profile, densities of ms were highest in the top 5 to 20 cm of soil but were consistently detected at 60-cm depths. Six weeks of natural flooding significantly reduced (on average, approximately 65% in the total viable counts of ms) but did not eliminate viable ms of V. longisporum.

Description of Cryptosporiopsis kienholzii and species profiles of Neofabraea in major pome fruit growing districts in the Pacific Northwest USA.

The objectives of this study were i) to give a taxonomic description of a fungus phylogenetically related to Neofabraea and assign the name Cryptosporiopsis kienholzii to this fungus, ii) to expand previous Neofabraea species profiles from infected apple and pear fruit collected from major pome fruit production districts in Oregon and Washington, and iii) to determine the sensitivity of Neofabraea alba, Neofabraea malicorticis, Neofabraea perennans, and C. kienholzii to a range of fungicides. A name is given herein to the anamorph of the fungus previously called 'Neofabraea sp. nov.' in the literature, and the fungus is designated as C. kienholzii. The teleomorph of this fungus has never been observed in vivo or in vitro. N. alba, N. perennans, and C. kienholzii accounted for 61.3, 35.6, and 3.1 %, respectively, of 608 isolates obtained from pear fruit and 6.0, 81.3, and 12.7 % of 150 isolates from apple fruit. N. alba was the most common species in Oregon and N. perennans was most common in Washington. N. malicorticis was not found in any of the districts and may be limited to the wet climates west of the Cascade mountain range. C. kienholzii occurs in pome fruit orchards from southern Oregon to North Central Washington, and this is the first report of C. kienholzii in the state of Washington. The four most effective fungicides for control of bull's-eye rot caused by all species were thiabendazole, thiophanate-methyl, pyrimethanil, and pyraclostrobin+boscalid. Other fungicides gave acceptable control of some species of Neofabraea but not others. There was good agreement of the microbioassay with fungicide evaluations using wound-inoculated pear fruit for five of seven fungicides, but the microbioassay was not consistent enough for determination of the sensitivity of Neofabraea species to new fungicides.