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Four species belonging to Colletotrichum acutatum and Colletotrichum gloeosporioides species complexes, including C. aenigma, C. fioriniae, C. fructicola, and C. nymphaeae, were identified from ripe rot symptomatic fruit from Mid-Atlantic vineyards. A quantitative real-time PCR (qPCR) SYBR Green method was developed to detect and quantify conidia of the two Colletotrichum complexes to better understand the distribution and the extent of Colletotrichum inoculum. Primers were designed to specifically target the β-tubulin gene of the C. acutatum complex or the C. gloeosporioides complex and were found to be specific to the desired complex, not amplifying DNA of other prevalent fungi from vineyards. The sensitivity of the primers was also assessed, and the C. acutatum complex-specific and C. gloeosporioides complex-specific primers were able to quantify as little as 50 C. fioriniae conidia and 100 C. aenigma conidia, respectively. Therefore, this would be a cost-effective, specific, and sensitive detection and quantification method for these two destructive species complexes in vineyards and many other crops through spore trapping applications.

Multiple Colletotrichum species have been found to be responsible for strawberry anthracnose, and prevalence of each species seems to vary by regions and/or host tissues. In this study, a total of 200 Colletotrichum isolates were obtained from different strawberry cultivars displaying anthracnose symptoms in the mid-Atlantic fields. Analysis of g3pdh, tub2, and/or internal transcribed spacer sequences revealed four Colletotrichum species, including C. nymphaeae, C. fioriniae, C. siamense, and C. lineola. C. nymphaeae was the predominant species, representing 90% of all isolates collected. This species was found from all strawberry organs/tissues examined, whereas C. siamense and C. fioriniae were limited to the crown and fruit, respectively. Further, all Colletotrichum isolates were screened for resistance to azoxystrobin in vitro, and all C. siamense isolates were additionally screened for resistance to thiophanate-methyl. The overall frequency of resistance to azoxystrobin and thiophanate-methyl was 48 and 67%, respectively. G143A in the cytochrome b gene was found in all C. nymphaeae and C. siamense isolates with high level of resistance, with EC50 >100 µg/ml, while F129L was found in two of the five C. nymphaeae isolates with moderate resistance, with EC50 values ranging from 2.6 to 7.8 µg/ml. All C. fioriniae isolates tested were found to be less sensitive to azoxystrobin, with EC50 values ranging from 9.7 to 14.4 µg/ml, despite no mutations detected in the cytochrome b gene. Moreover, E198A in tub2 was linked with C. siamense isolates resistant to thiophanate-methyl (EC50 >100 µg/ml). These results revealed that resistance in Colletotrichum spp. to primary fungicides is widespread in the mid-Atlantic strawberry fields.

Although Botrytis fragariae was only recently identified as a new Botrytis species that causes strawberry gray mold, its prevalence extends to many states of the eastern United States. Compared with B. cinerea, which is known to be the causal agent of gray mold on many crop plants including strawberry, B. fragariae appears to have specifically adapted to strawberry and exhibits distinct fungicide sensitivity. This is the first presentation of a high-quality genome assembly of B. fragariae with gene annotation based on sequence homology and deep transcriptome data. The genome sequence information from B. fragariae is expected to help reveal genomic features underlying its host specialization and evolution of distinct fungicide resistance and other novel pathogenicity mechanisms.

Horticultural performance of eleven blackberry cultivars, including two primocane-fruiting cultivars, was evaluated over three years after fruiting began at the Musser Fruit Research Center in Sout...

Colletotrichum spp. isolates contain two paralogous CYP51 genes that encode sterol 14-demethylase enzymes; however, their role in sensitivity to demethylation inhibitor (DMI) fungicides is yet to be determined. In this study, each of the two genes from Colletotrichum fioriniae and C. nymphaeae was able to rescue the function of CYP51 in the yeast Saccharomyces cerevisiae, demonstrating their independent function. Deletion of CYP51A led to increased sensitivity to propiconazole, diniconazole, prothioconazole, cyproconazole, epoxiconazole, flutriafol, prochloraz, and difenoconazole in C. fioriniae, and to the same fungicides and tebuconazole in C. nymphaeae, with the exception of prochloraz. Deletion of CYP51B in C. fioriniae and CYP51B in C. nymphaeae made mutants increasingly sensitive to five of nine DMI fungicides tested. The results suggest species-specific, differential binding of DMI fungicides onto the two CYP51 enzymes. Pairing DMIs with different effects on CYP51A and -B deletion mutants resulted in synergistic effects, as determined in mycelial growth inhibition experiments. Deletion mutants showed no fitness penalty in terms of mycelial growth, sporulation, and virulence. Our study elucidates the effect of CYP51A and CYP51B of Colletotrichum spp. on DMI sensitivity, suggesting that using a mixture of DMIs may improve the efficacy for anthracnose management.

Non-locality and quantum measurement are two fundamental topics in quantum theory and theirinterplay attracts intensive focus since the discovery of Bell theorem. Non-locality sharing amongmultiple observers is predicted and experimentally observed. However, only one-sided sequentialcase, i.e., one Alice and multiple Bobs is widely discussed and little is known about two-sided case.Here, we theoretically and experimentally explore the non-locality sharing in two-sided sequentialmeasurements case in which one entangled pair is distributed to multiple Alices and Bobs. Weexperimentally observed double EPR steering among four observers in the photonic system for thefirst time. In the case that all observers adopt the same measurement strength, it is observedthat double EPR steering can be demonstrated simultaneously while double Bell-CHSH inequalityviolations are shown to be impossible. The exact formula relating Bell quantity and sequential weakmeasurements for arbitrary many Alices and Bobs is also derived, showing that no more doubleBell-CHSH inequality violations is possible under unbiased input condition. The results not onlydeepen our understanding of relation between sequential measurements and non-locality but alsomay find important applications in quantum information tasks., Comment: 8 pages, 4 figures

Accurate control of qubits is the central requirement for building functional quantum processors. For the current superconducting quantum processor, high-fidelity control of qubits is mainly based on independently calibrated microwave pulses, which could differ from each other in frequencies, amplitudes, and phases. With this control strategy, the needed physical source could be challenging, especially when scaling up to large-scale quantum processors is considered. Inspired by Kane's proposal for spin-based quantum computing, here, we explore theoretically the possibility of baseband flux control of superconducting qubits with only shared and always-on microwave drives. In our strategy, qubits are by default far detuned from the drive during system idle periods, qubit readout and baseband flux-controlled two-qubit gates can thus be realized with minimal impacts from the always-on drive. By contrast, during working periods, qubits are tuned on resonance with the drive and single-qubit gates can be realized. Therefore, universal qubit control can be achieved with only baseband flux pulses and always-on shared microwave drives. We apply this strategy to the qubit architecture where tunable qubits are coupled via a tunable coupler, and the analysis shows that high-fidelity qubit control is possible. Besides, the baseband control strategy needs fewer physical resources, such as control electronics and cooling power in cryogenic systems, than that of microwave control. More importantly, the flexibility of baseband flux control could be employed for addressing the non-uniformity issue of superconducting qubits, potentially allowing the realization of multiplexing and cross-bar technologies and thus controlling large numbers of qubits with fewer control lines. We thus expect that baseband control with shared microwave drives can help build large-scale superconducting quantum processors., Comment: 16 pages, 16 figures

Resistance to fludioxonil in Botrytis cinerea and B. fragariae was previously found to be linked to either overexpression of the drug efflux pump atrB activated by mutations in transcription factor mrr1 or to mutations in the osmoregulation gene os1. In the present study, isolates of B. cinerea, Botrytis group S, or B. fragariae collected from strawberry fields in the United States were resistant to fludioxonil with half-maximal effective concentration values ranging from 0.04 to 0.43 µg/ml for B. cinerea, 0.03 to 1.03 µg/ml for Botrytis group S, and 0.28 to 3.48 µg/ml for B. fragariae. Analyses of mrr1 sequences revealed various mutations linked to fludioxonil resistance in B. cinerea and Botrytis group S isolates. However, no mutations in mrr1 correlated with atrB overexpression-mediated resistance in B. fragariae isolates. Neither nucleotide variations in the 1,370-bp upstream region of atrB nor increased atrB copy numbers could explain the atrB overexpression in these B. fragariae isolates. Mutations in os1 conferred resistance to iprodione in B. cinerea and Botrytis group S isolates; none correlated with resistance to fludioxonil in B. fragariae. In contrast to European isolates, U.S. B. fragariae isolates contained a 3-bp insertion in the coding region of os1. These isolates were more sensitive to osmotic stress but it is unclear whether the insertion is responsible for this phenotype. Our findings suggest that atrB overexpression-associated fludioxonil resistance is an across-species mechanism of resistance to fludioxonil that can be induced by mutations in mrr1 and other, still-unknown mechanisms.

From 2014 to 2019, 249 isolates of Botrytis spp. were collected from blackberry, black raspberry, grape, red raspberry, and strawberry showing gray mold symptoms. All isolates were phylogenetically characterized as Botrytis cinerea. A mycelial growth assay determined the following overall frequencies of resistance to fungicides: 92% to pyraclostrobin, 86% to cyprodinil, 71% to thiophanate-methyl, 48% to fenhexamid, 47% to iprodione, 26% to boscalid, 11% to fludioxonil, 8% to penthiopyrad, 7% to benzovindiflupyr, 4% to pydiflumetofen, and 4% to isofetamid. Isolates collected from blackberry, red raspberry, and strawberry had a higher median chemical class resistance value compared with isolates from black raspberry and grape. Resistance-conferring mutations were found in a selection of isolates characterized as resistant to thiophanate-methyl, iprodione, pyraclostrobin, fenhexamid, and boscalid, including E198A in β-tubulin; I365N/S, Q369P, and N373S in bos1; G143A in cytb; P238S, N369D, and F412I/S in erg27; and P225F and H272R/Y in sdhB, respectively. Also, multiple drug resistance (MDR) phenotypes MDR1 and MDR1h were identified by analyzing fludioxonil sensitivity and mrr1 sequences. MDR1 and MDR1h isolates had multiple amino acid variations and two insertions in mrr1 that resembled the group S genotype. A detached grape assay confirmed that the aforementioned mutations in isolates from different small fruit crops resulted in field-relevant resistance. An additional in vitro assay found that values for the effective concentration that inhibits mycelial growth by 50% of B. cinerea isolates to pydiflumetofen and inpyrfluxam averaged 0.4 and 1.0, 0.8 and 0.7, 149.8 and 23.2, 0.9 and 0.9, and 38.8 and 48.8 µg/ml for the wild-type, H272R, H272Y, N230I, and P225F genotypes, respectively. These results revealed widespread fungicide resistance in B. cinerea from Mid-Atlantic small fruit fields, highlighting the need for resistance management alternatives.

The rapid emergence of resistance in plant pathogens to the limited number of chemical classes of fungicides challenges sustainability and profitability of crop production worldwide. Understanding mechanisms underlying fungicide resistance facilitates monitoring of resistant populations at large-scale, and can guide and accelerate the development of novel fungicides. A majority of modern fungicides act to disrupt a biochemical function via binding a specific target protein in the pathway. While target-site based mechanisms such as alternation and overexpression of target genes have been commonly found to confer resistance across many fungal species, it is not uncommon to encounter resistant phenotypes without altered or overexpressed target sites. However, such non-target site mechanisms are relatively understudied, due in part to the complexity of the fungal genome network. This type of resistance can oftentimes be transient and noninheritable, further hindering research efforts. In this review, we focused on crop pathogens and summarized reported mechanisms of resistance that are otherwise related to target-sites, including increased activity of efflux pumps, metabolic circumvention, detoxification, standing genetic variations, regulation of stress response pathways, and single nucleotide polymorphisms (SNPs) or mutations. In addition, novel mechanisms of drug resistance recently characterized in human pathogens are reviewed in the context of nontarget-directed resistance.

Anthracnose fruit rot (AFR) and Botrytis fruit rot (BFR) are primary diseases affecting strawberry (Fragaria × ananassa), which typically drive fungicide applications throughout the growing season. The Strawberry Advisory System (StAS), a disease forecasting tool, was originally developed in Florida to better time the fungicide sprays by monitoring AFR and BFR infection risk based on leaf wetness and temperature input in real-time. Thirteen field trials were conducted in Maryland and Virginia between 2017 and 2019 to evaluate the StAS performance in the Mid-Atlantic region. As a result, 55, 18, and 31% fewer sprays were recorded on average in the model-based StAS treatment compared with the grower standard treatment in 2017, 2018, and 2019, respectively. Marketable yield, as well as AFR and BFR incidence, were largely comparable between the two treatments. However, poor disease control occurred during the StAS treatment in four trials in 2017, presumably because of a missed fungicide spray during a high-risk infection event and attributable to heavy rainfall that led to impassable fields. The implementation of the StAS may be further challenged by the employment of floating row covers that are essential for growing strawberries in plasticulture systems in open fields in the Mid-Atlantic region. Preliminary results indicated that row covers can alter canopy-level microclimatic conditions, possibly increasing the risk for disease occurrence. Overall, the StAS can be a valuable tool for Mid-Atlantic growers to control AFR and BFR, but sprays may need to be promptly applied when consecutive or heavy rainfalls are predicted, especially for highly susceptible cultivars. Complications in disease forecasting and management arising from the use of row covers need to be further addressed in this region because of its highly diverse climate.

The insulin superfamily is a group of homologous proteins that are further divided into the insulin family and relaxin family according to their distinct receptors. All insulin superfamily members contain three absolutely conserved disulfide linkages and a nonchiral Gly residue immediately following the first B-chain cysteine. The functionality of this conserved Gly residue in the insulin family has been studied by replacing it with natural L-amino acids or the corresponding unnatural D-amino acids. However, such analysis has not been conducted on relaxin family members. In the present study, we conducted chiral mutagenesis on the conserved B11Gly of the chimeric relaxin family peptide R3/I5, which is an efficient agonist for receptor RXFP3 and RXFP4. Similar to the effects on insulin family foldability, L-Ala or L-Ser substitution completely abolished the in vitro refolding of a recombinant R3/I5 precursor; whereas, D-Ala or D-Ser substitution had no detrimental effect on refolding of a semi-synthetic R3/I5 precursor, suggesting that the conserved Gly residue controls the foldability of relaxin family members. In contrast to the effect on insulin family activity, D-Ala or D-Ser replacement had no detrimental effect on the binding and activation potencies of the mature R3/I5 towards both RXFP3 and RXFP4, suggesting that the conserved Gly residue is irrelevant to the relaxin family's activity. The present study revealed functionality of the conserved B-chain Gly residue for a relaxin family peptide for the first time, providing an overview of its contribution to foldability and activity of the insulin superfamily.

Relaxin family peptides perform a variety of biological functions by binding and activating relaxin family peptide receptor 1-4 (RXFP1-4), four A-class G protein-coupled receptors. In the present work, we developed a novel ligand binding assay for RXFP3 and RXFP4 based on NanoLuc complementation technology (NanoBiT). A synthetic ligation version of the low-affinity small complementation tag (SmBiT) was efficiently ligated to the A-chain N terminus of recombinant chimeric agonist R3/I5 using recombinant circular sortase A. After the ligation product R3/I5-SmBiT was mixed with human RXFP3 or RXFP4 genetically fused with a secretory large NanoLuc fragment (sLgBiT) at the N terminus, NanoLuc complementation was induced by high-affinity ligand-receptor binding. Binding kinetics and affinities of R3/I5-SmBiT with sLgBiT-fused RXFP3 and RXFP4 were conveniently measured according to the complementation-induced bioluminescence. Using R3/I5-SmBiT and the sLgBiT-fused receptor as a complementation pair, binding potencies of various ligands with RXFP3 and RXFP4 were quantitatively measured without the cumbersome washing step. The novel NanoBiT-based ligand binding assay is convenient for use and suitable for automation, thus will facilitate interaction studies of RXFP3 and RXFP4 with ligands in future. This assay can also be applied to some other plasma membrane receptors for pharmacological characterization of ligands in future studies.

Relaxin family is a group of peptide hormones with a variety of biological functions by activating G protein-coupled receptors RXFP1–4. We recently developed bioluminescent tracers for their receptor-binding assays by chemical conjugation with the ultrasensitive NanoLuc reporter. To simplify preparation of the bioluminescent tracers, in the present study, we established a sortase-catalysed ligation approach using the chimeric R3/I5 as a model. Following catalysis by recombinant sortase A, a NanoLuc reporter carrying the LPETG sortase recognition motif at the C-terminus was efficiently ligated to an R3/I5 peptide carrying four successive Gly residues at the A-chain N-terminus, via the formation of a peptide bond between the C-terminal LPET sequence of NanoLuc and the A-chain N-terminal Gly residue of R3/I5. Saturation binding assays demonstrated that the NanoLuc-ligated R3/I5 retained high binding affinity to RXFP3 and RXFP4, with the calculated dissociation constants (K d) of 4.34 ± 0.33 nM (n = 3) and 5.66 ± 0.54 nM (n = 3), respectively. Using the NanoLuc-ligated R3/I5 as a tracer in competition binding assays, binding potencies of various ligands towards RXFP3 and RXFP4 were conveniently quantified. This work provides a simple method for rapid preparation of bioluminescent tracers for relaxin family peptides and other protein/peptide hormones for ligand–receptor interaction studies.

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely RXFP1–4. Among these receptors, RXFP3 lacks a specific natural or synthetic agonist at present. A previously designed chimeric R3/I5 peptide, consisting of the B-chain of relaxin-3 and the A-chain of INSL5, displays equal activity towards the homologous RXFP3 and RXFP4. To increase its selectivity towards RXFP3, in the present study we conducted extensive mutagenesis around the B-chain C-terminal region of R3/I5. Decreasing or increasing the peptide length around the B23–B25 position dramatically lowered the activation potency of R3/I5 towards both RXFP3 and RXFP4. Substitution of B23Gly with Ala or Ser converted R3/I5 from an efficient agonist to a strong antagonist for RXFP3, but the mutants retained considerable activation potency towards RXFP4. Substitution of B24Gly increased the selectivity of R3/I5 towards RXFP3 over the homologous RXFP4. The best mutant, [G(B24)S]R3/I5, displayed 20-fold higher activation potency towards RXFP3 than towards RXFP4, meanwhile retained full activation potency at RXFP3. Thus, [G(B24)S]R3/I5 is the best RXFP3-selective agonist known to date. It is a valuable tool for investigating the physiological functions of RXFP3, and also a suitable template for developing RXFP3-specific agonists in future.

Wavefunction is the foundation of quantum theory, which is assumed to give a complete description of a quantum system. For a long time, wavefunction is introduced as an abstract element of the theory and there lacks effective ways to measure it directly. The situation, however, is somewhat changed when Lundeen et al. reported the direct measurement of the quantum wavefunction via weak measurements, which gives the wavefunction a clearly operational definition [Nature 474, 188 (2011)]. The weak measurement method requires sequential measurements of conjugate observables position and momentum with the position measurement is weak enough. Surprisingly, the recent research by Vallone and Dequal shows that performing sequential strong measurements realizes the same target, in which case no approximation has to be made compared to the case of weak measurements[Phys. Rev. Lett. 116, 040502 (2016)]. Here we experimentally report the direct measurement of the two-dimensional transverse wavefunction of photons via strong measurements for the first time, which implies that an accurate and clear operational definition can be given to wavefunction. We have measured the Gaussian and Laguerre-Gaussian of l = 1 spatial wavefunctions of photons with R-square are 0.97 and 0.93 respectively. As a potentially important application, we show that the direct measurement of two-dimensional wavefunction provides an alternative way to realize digital holography of three-dimensional objects. The results presented here will not only deepen our understanding of abstract wavefunction but also have significant applications in quantum information processing and quantum imaging., 5 pages

Sequential weak measurements of non-commuting observables is not only fundamentally interesting in quantum measurement but also shown potential in various applications. The previous reported methods, however, can only realize limited sequential weak measurements experimentally. In this Letter, we propose the realization of sequential measurements of arbitrary observables and experimentally demonstrate for the first time the measurement of sequential weak values of three non-commuting Pauli observables by using genuine single photons. The results presented here will not only improve our understanding of quantum measurement, e.g. testing quantum contextuality, macroscopic realism, and uncertainty relation, but also have many applications such as realizing counterfactual computation, direct process tomography, direct measurement of the density matrix and unbounded randomness certification., Comment: Comments are welcome!

In 2009 and 2010, peaches (Prunus persica) with brown rot symptoms were collected from Zhejiang Plant Protection State Research Farm and a commercial orchard in Fujian Province in southeastern China. Affected fruit showed brown decay with zones of sporulation. Single-spore isolates from the diseased fruit were cultured on potato dextrose agar. After incubation at 25°C in the dark for 5 days, colonies were gray with concentric rings of sporulation. Mean mycelial growth of isolates MZ09-2a from Zhejiang Province and 0907-a from Fujian Province was 4.46 ± 0.58 and 7.05 ± 0.81 cm after 4 and 7 days of incubation, respectively. Lemon-shaped conidia were formed in branched, monilioid chains and mean size was 14.6 (9.6 to 21.6) × 10.3 (7.2 to 13.2) μm. Mean conidial germination was 97 ± 1% with one straight germ tube per conidium. These characteristics were consistent with descriptions of Monilinia fructicola (G. Wint.) Honey (3). Morphology-based species identification was confirmed by sequencing and analysis of ribosomal internal transcribed spacer (ITS) sequences. A 496-bp fragment including ITS 1 and 2 and the gene encoding the 5.8S small subunit of the ribosomal RNA from isolates MZ09-2a and 0907-a was amplified using the universal primer pair ITS1/ITS4 (4) and sequenced. Nucleotide sequences of both isolates were identical. Blast searches of the ITS sequences in GenBank showed the highest similarity (100%) with sequences of M. fructicola isolates from China (FJ515894), Italy (FJ411109), and Spain (EF207423). The isolates were also identified as M. fructicola using the Monilinia spp. PCR detection protocol based on sequence-characterized amplification region marker DNA sequences (2). Pathogenicity was confirmed by inoculating surface-sterilized, mature cv. Zhonghua 2 peaches with mycelial plugs of representative isolates. Fruit was stabbed at two points with a 5-mm-diameter sterile cork borer, mycelial plugs (5 mm in diameter) were removed from the periphery of a 4-day-old colony of each isolate and placed upside down into each wound; control fruit received water agar. Inoculated fruit developed typical brown rot symptoms with sporulating fungi while control fruit remained healthy after 3 days of incubation at 22°C in a moist chamber. Pathogens were reisolated from the inoculated fruit and confirmed to be M. fructicola on the basis of morphological characteristics. To our knowledge, this is the first report of M. fructicola in Zhejiang and Fujian provinces. Both provinces are located more than 1,000 km south of Beijing, Hebei, and Shandong provinces, where M. fructicola had been reported previously (1). References: (1) J. Y. Fan et al. Acta Phytophylacica Sin. (in Chinese) 34:289, 2007. (2) I. Gell et al. J. Appl. Microbiol. 103:2629, 2007. (3) G. C. M. van Leeuwen and H. A. van Kesteren. Can. J. Bot. 76:2041, 1998. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds., Academic Press, San Diego, 1990.

Colletotrichum gloeosporioides, C. acutatum, and C. truncatum are causal agents of anthracnose disease of peach in South Carolina, but more recent investigations show that C. gloeosporioides is a species complex that has not yet been investigated among peach isolates. A total of 28 Colletotrichum spp. isolates associated with peach fruit anthracnose were collected in 2012 from Chesnee (10 isolates), McBee (10 isolates), Monetta (2 isolates), and Ridge Spring (6 isolates), South Carolina. Morphological characteristics indicated that all 28 isolates belonged to the C. gloeosporioides species complex. Phylogenetic analysis of the combined calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin (TUB2) gene sequences identified two species, C. siamense and C. fructicola. Cultural characteristics such as colony growth rate, shape and size of conidia, and appressoria from representative isolates of the two species largely matched previous descriptions for C. siamense and C. fructicola. Koch’s postulates for C. siamense and C. fructicola were fulfilled, confirming pathogenicity of the two species on peach. A new, two-step multiplex PCR assay was developed to facilitate differentiation of the four known Colletotrichum spp. causing anthracnose of peach in South Carolina, including C. acutatum, C. truncatum, C. siamense, and C. fructicola. The first step distinguished C. acutatum from C. truncatum and the two members of the C. gloeosporioides species complex. The second assay distinguished C. siamense from C. fructicola isolates.

Few fungicides are effective against anthracnose, caused by Colletotrichum spp., and emerging resistance makes the search for chemical alternatives more relevant. Isolates of the Colletotrichum acutatum species complex were collected from South Carolina and Georgia peach orchards and phylogenetic analysis of the combined internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase, and β-tubulin gene sequences separated the isolates into C. nymphaeae and C. fioriniae. The sensitivity of these and three other previously reported Colletotrichum spp. from peach, including C. fructicola, C. siamense, and C. truncatum, to demethylation inhibitor (DMI) fungicides difenoconazole, propiconazole, tebuconazole, metconazole, flutriafol, and fenbuconazole was determined based upon mycelial growth inhibition. C. truncatum was resistant to tebuconazole, metconazole, flutriafol, and fenbuconazole and C. nymphaeae was resistant to flutriafol and fenbuconazole based on 50% effective concentration (EC50) values >100 μg/ml. C. fructicola and C. siamense were sensitive to all DMI fungicides (EC50 values of 0.2 to 13.1 μg/ml). C. fioriniae subgroup 2 isolates were less sensitive to DMI fungicides (EC50 values of 0.5 to 16.2 μg/ml) compared with C. fioriniae subgroup 1 (EC50 values of 0.03 to 2.1 μg/ml). Difenoconazole and propiconazole provided the best control efficacy in vitro to all five species, with EC50 values of 0.2 to 2.7 μg/ml. Tebuconazole and metconazole were effective against all Colletotrichum spp., except for C. truncatum. The strong in vitro activity of some DMI fungicides against Colletotrichum spp. may be exploited for improved anthracnose disease management of peach.

Resistance to multiple chemical classes of fungicides in Botrytis cinerea isolates from eastern United States strawberry fields is common and strategies to control them are needed. In this study, we compared fitness and competitive ability of eight sensitive isolates (S), eight isolates resistant to five or six chemical classes of fungicides but not to phenylpyrroles (5CCR), and eight isolates resistant to six or seven chemical classes including phenylpyrroles (6CCR/MDR1h). The latter included the MDR1h phenotype due to overexpression of atrB based on Δ497V/L in mrr1. The 6CCR/MDR1h isolates grew more slowly at 4°C on potato dextrose agar, and both 5CCR and 6CCR/MDR1h isolates were hypersensitive to osmotic stress compared with S isolates. In contrast, no differences were found in oxidative sensitivity, aggressiveness, and spore production in vivo, and sclerotia production and viability in vitro. In competition experiments, the 5CCR and 6CCR/MDR1h isolates were both outcompeted by S isolates and 6CCR/MDR1h isolates were outcompeted by 5CCR isolates in the absence of fungicide pressure. Under selective pressure of a fludioxonil/pyraclostrobin rotation, the 6CCR/MDR1h isolates dominated after coinoculation with 5CCR and S isolates. The competitive disadvantage of 5CCR and especially 6CCR/MDR1h isolates suggest that, in the absence of fungicide selection pressure, S isolates may reduce inoculum potential of multifungicide-resistant isolates under field conditions.

Relaxin-3 is an insulin/relaxin superfamily neuropeptide implicated in the regulation of food intake and stress response via activation of the G protein-coupled receptor RXFP3. Their electrostatic interactions have been recently identified, and involves three positively charged B-chain residues (B12Arg, B16Arg, and B26Arg) of relaxin-3 and two negatively charged residues (Glu141 and Asp145) in a highly conserved ExxxD motif at the extracellular end of the second transmembrane domain of RXFP3. To investigate their hydrophobic interactions, in the present work we deleted the highly conserved B-chain C-terminal B27Trp residue of relaxin-3, and mutated four highly conserved aromatic residues (Phe137, Trp138, Phe146, and Trp148) around the ExxxD motif of RXFP3. The resultant [∆B27W]relaxin-3 exhibited approximately tenfold lower binding potency and ~1000-fold lower activation potency towards wild-type RXFP3, confirming its importance for relaxin-3 function. Although the RXFP3 mutants could be normally trafficked to cell membrane, they had quite different activities. [F137A]RXFP3 could normally distinguish wild-type relaxin-3 and [∆B27W]relaxin-3 in binding and activation assays, whereas [W138A]RXFP3 lost most of this capability, suggesting that the Trp138 residue of RXFP3 forms hydrophobic interactions with the B27Trp residue of relaxin-3. The hydrophobic Trp138 residue and the formerly identified negatively charged Glu141 and Asp145 residues in the highly conserved WxxExxxD motif may thus form a functional surface that is important for interaction with relaxin-3. We hypothesize that the relaxin-3 B-chain C-terminus changes from the original folding-back conformation to an extended conformation during binding with RXFP3, to allow its B27Trp and B26Arg residues to interact with the Trp138 and Glu141 residues of RXFP3, respectively.

Succinate dehydrogenase inhibitor (SDHI) fungicides have been used to control gray mold of strawberry for more than a decade, and selection for resistance in the causal agent Botrytis cinerea has become a threat to producers. In total, 2,570 B. cinerea isolates were collected from strawberry fields in the eastern United States across three seasons and their sensitivity to the SDHI materials boscalid, fluopyram, fluxapyroxad, and penthiopyrad was assessed. Assays were based on visual assessment of presence or absence of mycelial growth on media amended with discriminatory fungicide doses to distinguish sensitive from resistant isolates, respectively. Overall frequencies of isolates resistant to boscalid, fluopyram, fluxapyroxad, and penthiopyrad increased over the 3 years to 30.0, 1.0, 5.5, and 7.4%, respectively. Four resistance patterns, designated A, B, C, or D, were found. Pattern A isolates were resistant to boscalid with the allele H272R at locus sdhB; pattern B isolates were resistant to boscalid and penthiopyrad with the allele H272R or H272Y at locus sdhB; pattern C isolates were resistant to boscalid, fluxapyroxad, and penthiopyrad with the allele H272Y at locus sdhB; and pattern D isolates were resistant to boscalid, fluopyram, fluxapyroxad, and penthiopyrad with alleles P225F or N230I at locus sdhB. Isolates with alleles H272Y, N230I, or P225F were sensitive to a new SDHI, benzovindiflupyr, with mean effective dose that inhibits 50% of mycelial growth values of less than 0.5 μg/ml for each genotype, suggesting that this fungicide may be useful for resistance management. Our data show an increase of B. cinerea isolates resistant to SDHI fungicides over three consecutive production seasons. Resistance management practices must be implemented for the sustained efficacy of SDHI fungicides against gray mold of strawberry.

Colletotrichum fioriniae Marcelino & Gouli ex R.G. Shivas & Y.P. Tan causes anthracnose and fruit rot in various hosts worldwide (Shivas and Tan 2009). In June 2019, 40% of red raspberry fruit (cv. Prelude) in a plot at the Wye Research and Education Center of the University of Maryland in Queenstown, MD, showed light to dark brown, rotten, and slightly sunken drupelets. Symptomatic berries (n = 17) were collected and incubated in plastic boxes at room temperature (22°C) to obtain 41 single-spore isolates. Colonies on potato dextrose agar (PDA) were gray to pale olivaceous, cottony, with aerial mycelium in compact tufts, and had a white rim and a pinkish-red to pale orange or yellow reverse and orange acervuli. Conidia were one-celled, hyaline, smooth-walled, with either both acute ends or one round and one pointed end, without setae. Conidial dimensions were recorded for two single-spore isolates and were in accordance with previous measurements (Damm et al. 2012): mean length ± SD × width ± SD = 12.9 ± 1.5 × 5.4 ± 0.9 μm on PDA (n = 60). DNA was extracted from mycelium of 11 single-spore isolates (Chi et al. 2009). Damm et al. (2012) showed that all subclades of the Colletotrichum acutatum complex can be identified by amplifying a 290-bp section of the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene (primers: Templeton et al. [1992]) and a 1,440-bp section of the β-tubulin-2 gene (tub2) (primers: T1 from O’Donnell and Cigelnik [1997], TubR1 from Hu et al. [2015]). Polymerase chain reaction products were sequenced from two directions. Three consensus sequences for each gene were deposited into GenBank: RB19-14.4S (MN520488, MN532086), RB19-13.3S (MN520489, MN532087), and RB19-26P (MN520490, MN532088). BLASTn search confirmed 100% identity with the Colletotrichum fioriniae sequences JQ949974 (491 bp, tub2) and JQ948653 (254 bp, gapdh) for RB19-14.4S and in total nine of the 11 isolates. For isolate RB19-26P the gapdh sequence (MN532088) was 100% identical with JQ948624 instead. Tub2 (MN520489) and gapdh (MN532087) sequences for isolate RB19-13.3S were 100 and 99.6% identical with C. fioriniae sequences JQ949944 and JQ948623, respectively. To confirm pathogenicity, healthy ripe red raspberries (cv. Jaclyn) were inoculated with a mixture of four single-spore isolates. Berries were surface sterilized by washing in 0.1% Tween 20 for 2 min, 70% ethanol for 30 s, 1% NaOCl for 1 min, and sterile water for 2 min. After air drying for 2 h, berries were either wounded with a sterile toothpick or not wounded and were inoculated with a 15-μl drop of spore suspension (5 × 10⁵ conidia/ml) or sterile water. Each treatment was replicated four times with eight berries per replicate. The experiment was conducted twice. After incubation for 3 days, both wounded and unwounded inoculated berries showed symptoms identical to those described above, and some lesions had orange acervuli. C. fioriniae was reisolated from inoculated berries but never from water-inoculated berries. Identity of the recovered isolates was confirmed morphologically and molecularly. Although C. fioriniae is an endophyte in various plant species worldwide (Freeman et al. 2001) and was previously isolated as an endophyte or from leaves of Rubus idaeus in New York (U.S.A.), Norway, and Mississippi (U.S.A.) (Marcelino et al. 2009; Sundelin et al. 2015; Zhang et al. 2016), it has not been described as a raspberry fruit pathogen causing anthracnose disease so far. To our knowledge this is the first report of C. fioriniae found on red raspberries in the Mid-Atlantic region of the United States.

Polyoxin D is a Fungicide Resistance Action Committee (FRAC) code 19 fungicide that was recently registered for gray mold control of strawberry in the United States. In this study, we determined the sensitivity to polyoxin D zinc salt (hereafter, polyoxin D) of Botrytis cinerea isolates from 41 commercial strawberry farms in South Carolina, North Carolina, Maryland, Virginia, and Ohio and investigated the fitness of sensitive (S) and reduced sensitive (RS) isolates. Relative mycelial growth ranged between 0 and over 100% on malt extract agar amended with a discriminatory dose of polyoxin D at 5 μg/ml. Isolates that grew more than 70% at that dose were designated RS and were found in three of the five states. The 50% effective dose (EC50) values of three S and three RS isolates ranged from 0.59 to 2.27 and 4.6 to 5.8 μg/ml, respectively. The three RS isolates grew faster on detached tomato fruit treated with Ph-D WDG at recommended label dosage than S isolates (P < 0.008). In all, 25 randomly selected RS isolates exhibited reduced sporulation ability (P < 0.0001) and growth rate (P < 0.0001) but increased production of sclerotia (P < 0.0386) compared with 25 S isolates. Of 10 isolates tested per phenotype, the number of RS isolates producing sporulating lesions on apple, tomato, and strawberry was significantly lower compared with S isolates (P < 0.0001 for each fruit type). The results of this study indicate that resistance management is necessary for fungicides containing polyoxin D. To our knowledge, this is the first study demonstrating reduced sensitivity to FRAC 19 fungicides in B. cinerea isolates from the United States.

Gray mold is a devastating disease on strawberry, and may be caused by several species of Botrytis. The goal of this study was to better understand and characterize the species of Botrytis with reduced sensitivity to the fungicide Polyoxin D, particularly Botrytis fragariae. In total, 78 Botrytis isolates of unknown species that were sensitive (28 isolates; S), moderately sensitive (22 isolates; MS), or reduced sensitive (28 isolates; RS) to Polyoxin-D were collected from commercial strawberry fields of five states in the United States, identified to the species level, and characterized. The majority (75%) of S isolates were Botrytis cinerea and the majority (79%) of RS isolates were the recently described species B. fragariae, indicating an innate ability of B. fragariae to tolerate Polyoxin-D. B. fragariae produced fluffy, white mycelium and was less likely to sporulate on potato dextrose agar than B. cinerea. Isolates from a commercial field recovered from blossoms in early spring were all B. fragariae, those from leaves of the same plants in late spring were a mixture of B. fragariae and B. cinerea, and those from fruit in early summer were all B. cinerea, indicating that B. fragariae may preferentially colonize blossom tissue. A polymerase chain reaction-based assay was developed based on NEP2 sequence variability to distinguish B. fragariae from other Botrytis spp. that have been reported on strawberry, including B. cinerea, B. mali, B. caroliniana, and B. ricini. None of the isolates collected from Canada, California, or North Carolina nurseries were B. fragariae, indicating that the newly described species may not exist or not be widely distributed in planting stock.

Gray mold, caused by Botrytis spp., is among the most devastating diseases affecting strawberry worldwide. The great diversity present in the pathogen enhances its ability to survive and adapt in the field. In this study, we explored the genotypic and phenotypic diversity present in single strawberry flowers. In total, 192 isolates were collected from 19 flowers and four farms, and 9 to 12 isolates were collected from each flower. Forty-two haplotypes were found using microsatellite fragment analysis. Multiple haplotypes of two different Botrytis spp. (Botrytis cinerea and B. fragariae) were found in 12 flowers. In the remaining seven flowers, the single-spore isolates examined were of identical haplotypes. In three flowers, the two Botrytis spp. were found to coexist. Isolates were either sensitive (zero chemical class resistance) or resistant to one, two, three, four, or five chemical classes of fungicides. Resistance to multiple fungicides was commonly observed in both species but resistance to boscalid and penthiopyrad was only found in B. cinerea isolates. Resistance to cyprodinil was found in B. fragariae for the first time in the United States. Each haplotype was generally linked to a single resistance profile; however, a single resistance profile often was represented by multiple haplotypes. Isolates from the same flower of multiple haplotypes were largely identical in resistance profiles. This study is a first detailed investigation of genotypic diversity combined with phenotypic analysis of Botrytis spp. at the single-tissue level. It demonstrates that high genotypic and phenotypic diversity is present not only within fields but also in individual blossoms as well. This information is important for understanding the epidemiology of Botrytis and also has implications for fungicide resistance management, particularly related to resistance monitoring practices.

We experimentally realize the internal and external entanglement tradeoff, which is a new kind of entanglement monogamy relation different from that usually discussed. Using a source of twin photons, we find that the external entanglement in polarization of twin photons, and the path-polarization internal entanglement of one photon, limit each other. In the extreme case, when the internal state is maximally entangled, the external entanglement must be vanishing, that illustrate entanglement monogamy. Our results of the experiment coincide with the theoretical predictions, and therefore provide a direct experimental observation of the internal and external entanglement monogamy relation., 10 pages, 7 figures

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely relaxin family peptide receptor 1-4 (RXFP1-4). We recently disclosed electrostatic interactions of the homologous RXFP3 and RXFP4 with some agonists based on activation complementation. However, this activation assay-based approach cannot be applied to antagonists that do not activate receptors. Herein, we propose a general approach suitable for both agonists and antagonists based on our newly-developed NanoBiT-based binding assay. We first validated the binding assay-based approach using the agonist relaxin-3, then applied it to the chimeric antagonist R3(ΔB23-27)R/I5. Three positively charged B-chain Arg residues of the agonist and antagonist were respectively replaced by a negatively charged Glu residue; meanwhile, the negatively charged Glu and Asp residue in the essential WxxExxxD motif of both receptors were respectively replaced by a positively charged Arg residue. Based on binding complementation of mutant ligands towards mutant receptors, we deduced possible electrostatic interactions of the agonist and antagonist with both RXFP3 and RXFP4: their B-chain C-terminal Arg residue interacts with the deeply buried Glu residue in the WxxExxxD motif of both receptors, and one or two of their B-chain central Arg residues interact with the shallowly buried Asp residue in the WxxExxxD motif of both receptors. Our present work shed new light on the interaction mechanism of RXFP3 and RXFP4 with agonists and antagonists, and also provided a novel approach for interaction studies of some plasma membrane receptors with their ligands.

Anthracnose fruit rot was observed in some late-season peach cultivars in South Carolina in the 2012 and 2013 production seasons as well as increased anthracnose leaf spot of blueberry in a commercial operation of the same state in 2012. Single-spore isolates of Colletotrichum siamense were either sensitive or resistant to both thiophanate-methyl and azoxystrobin with the concentration of the fungicide at which fungal development is inhibited by 50% of ≥100 μg/ml. Resistant isolates revealed the E198A mutation in β-tubulin and the G143A mutation in cytochrome b. Nucleotide sequence analysis of the complete CYTB gene from genomic DNA of C. siamense isolates revealed an intronless genotype (CsI) and a genotype revealing two introns (CsII) at amino acid positions 131 and 164. Resistance to thiophanate-methyl or azoxystrobin was not found in isolates of C. fructicola collected from peach fruit. The CYTB gene of isolates of this species was of the CfII genotype or revealed a unique CfIIa genotype. Phylogenetic analysis of C. siamense isolates from different locations and different crops showed that the resistant isolates were genetically closer to each other than to sensitive isolates, suggesting that field resistance to thiophanate-methyl and azoxystrobin fungicides is derived from a common ancestor.

We experimentally test the recently predicted anisotropic invariance properties of pure three-qubit states, via generation and measurement of polarisation-path entangled three-qubit states. These properties do not require aligned reference frames and can be determined from measurements on any two of the qubits. They have several applications, such as a universal ordering of pairwise quantum correlations, strong monogamy relations for Bell inequalities and quantum steering, and a complementarity relation for Bell nonlocality versus 3-tangle, some of which we also test. The results indicate that anisotropic invariance, together with the three qubit Bloch vector lengths, can provide a robust and complete set of invariants for such states under local unitary transformations., Comment: 6 pages, 1 figure

Relaxin family peptide receptor 3 (RXFP3) is an A-class G protein-coupled receptor that is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. To study its interaction with various ligands, we developed a novel bioluminescence resonance energy transfer (BRET)-based binding assay using the brightest NanoLuc as an energy donor and a newly developed cyan-excitable orange fluorescent protein (CyOFP) as an energy acceptor. An engineered CyOFP without intrinsic cysteine residues but with an introduced cysteine at the C-terminus was overexpressed in Escherichia coli and chemically conjugated to the A-chain N-terminus of an easily labeled chimeric R3/I5 peptide via an intermolecular disulfide linkage. After the CyOFP-conjugated R3/I5 bound to a shortened human RXFP3 (removal of 33 N-terminal residues) fused with the NanoLuc reporter at the N-terminus, high BRET signals were detected. Saturation binding and real-time binding assays demonstrated that this BRET pair retained high binding affinity with fast association/dissociation. Using this BRET pair, binding potencies of various ligands with RXFP3 were conveniently measured through competition binding assays. Thus, the novel BRET-based binding assay facilitates interaction studies of RXFP3 with various ligands. The engineered CyOFP without intrinsic cysteine residues may also be applied to other BRET-based binding assays in future studies.

As one of the most intriguing intrinsic properties of quantum world, quantum superposition provokes great interests in its own generation. Oszmaniec [Phys. Rev. Lett. 116, 110403 (2016)] have proven that though a universal quantum machine that creates superposition of arbitrary two unknown states is physically impossible, a probabilistic protocol exists in the case of two input states have nonzero overlaps with the referential state. Here we report a heralded quantum machine realizing superposition of arbitrary two unknown photonic qubits as long as they have nonzero overlaps with the horizontal polarization state $|H\rangle$. A total of 11 different qubit pairs are chosen to test this protocol by comparing the reconstructed output state with theoretical expected superposition of input states. We obtain the average fidelity as high as 0.99, which shows the excellent reliability of our realization. This realization not only deepens our understanding of quantum superposition but also has significant applications in quantum information and quantum computation, e.g., generating non-classical states in the context of quantum optics and realizing information compression by coherent superposition of results of independent runs of subroutines in a quantum computation., 4 pages, 2 figures

Previous research has shown that Botrytis cinerea isolates with resistance to multiple chemical classes of fungicides exist in eastern strawberry fields. In this study, the fungicide resistance profiles of 2,130 isolates from flowers of commercial strawberry fields located in multiple states was determined over four consecutive strawberry production seasons. Producers were asked to alternate single-site fungicides that were considered low risk in their specific location based on resistance monitoring results in their fields. This recommendation led to an increase of chemical class diversity used in the spray programs. Results indicated that simultaneous resistance in individual isolates to two, three, four, five, six, and seven classes of fungicides increased over time. The increase in chemical class resistances within isolates was likely due to a process we termed “selection by association”, where fungicide resistance traits were often linked to the trait being selected rather than the selectable trait itself. Data analysis also indicated that the odds were highest for isolates resistant to one chemical class (1CCR) to be resistant to thiophanate-methyl; for 2CCR isolates to be resistant to thiophanate-methyl and pyraclostrobin; and for 3CCR isolates to be resistant to thiophanate-methyl, pyraclostrobin, and either cyprodinil or fenhexamid. We hypothesize that the more chemical classes are used in a spray program, the faster isolates will be selected with increasing numbers of chemical class resistances by virtue of selection by association if such isolates preexist in the population.

The relaxin family peptides play a variety of biological functions by activating four G protein-coupled receptors, RXFP1–4. Among them, insulin-like peptide 5 (INSL5) and relaxin-3 share the highest sequence homology, but they have distinct receptor preference: INSL5 can activate RXFP4 only, while relaxin-3 can activate RXFP3, RXFP4 and RXFP1. Previous studies suggest that the A-chain is responsible for their different selectivity for RXFP1. However, the mechanism by which INSL5 distinguishes the homologous RXFP4 and RXFP3 remains unknown. In the present work, we chemically evolved INSL5 in vitro to a strong agonist of both RXFP4 and RXFP3 through replacement of its five B-chain residues with the corresponding residues of relaxin-3. We identified four determinants (B2Glu, B9Leu, B17Tyr and a rigid B-chain C-terminus) on INSL5 that are responsible for its inactivity at RXFP3. In reverse experiments, we grafted these determinants onto a chimeric R3/I5 peptide, which contains the B-chain of relaxin-3 and the A-chain of INSL5 and retains full activation potency at RXFP3 and RXFP4. All resultant R3/I5 mutants retained high activation potency towards RXFP4, but most displayed significantly decreased or even abolished activation potency towards RXFP3, confirming the role of these four INSL5 determinants in distinguishing RXFP4 from RXFP3.

Bell nonlocality plays a fundamental role in quantum theory. Numerous tests of the Bell inequality have been reported since the ground-breaking discovery of the Bell theorem.Up to now, however, most discussions of the Bell scenario have focused on a single pair of entangled particles distributed to only two separated observers. Recently, it has been shown surprisingly that multiple observers can share the nonlocality present in a single particle from an entangled pair using the method of weak measurements [Phys. Rev. Lett. {\bf 114}, 250401 (2015)]. Here we report an observation of double CHSH-Bell inequality violations for a single pair of entangled photons with strength continuous-tunable optimal weak measurements in photonic system for the first time. Our results not only shed new light on the interplay between nonlocality and quantum measurements but may also be significant for important applications such as unbounded randomness certification and quantum steering., Comment: 6 pages, 3 figures, close to publish version, accepted by njp Quantum Information

A universal deterministic noiseless quantum amplifier has been shown to be impossible. However, probabilistic noiseless amplification of a certain set of states is physically permissible. Regarding quantum state amplification as quantum state transformation, we show that deterministic noiseless amplification of coherent states chosen from a proper set is possible. The relation between input coherent states and gain of amplification for deterministic noiseless amplification is thus derived. Besides, the potential applications of amplification of coherent states in quantum key distribution (QKD), noisy channel and non-ideal detection are also discussed., Comment: 4 pages, 1 figure