Overcoming Dormancy in Prunus Species under Conditions of Insufficient Winter Chilling in Israel.

The phenomenon of dormancy and the evolutionary causes for its development are presented together with the effects of the climatic factors: temperature and light. Shade and darkness have been found to enhance bud breaking in peach. The effects of various temperatures on chilling accumulation, chilling negation and chilling enhancement are described. The way these are computed in the face of global warming is explained, using the dynamic model. When natural chilling is less than that required, there are ways of compensation, up to a certain level. Various horticultural, physical and chemical means to achieve this are described, including bending branches, reducing vegetative vigor, shading the orchard, sprinkling to reduce daytime temperature and the application of various chemicals to break dormancy. When winter chilling is markedly reduced and temperatures increase considerably, the use of dormancy avoidance is suggested in frost-free places. This technique can induce a new growing cycle by avoiding dormancy altogether. However, the best approach is to breed high-quality cultivars requiring much less chilling. Another aspect discussed in this work, independent of the chilling requirement, is the negative effect of heat spells in winter and spring on the abnormal development of flower buds, leading to a low level of the stone fruit set and a reduced yield.

Molecular Identification and Pathogenicity of Diaporthe eres and D. hongkongensis (Diaporthales, Ascomycota) Associated with Cherry Trunk Diseases in China.

This study aimed to identify fungal species associated with trunk diseases of sweet cherries (Prunus avium) in several commercial cherry orchards in Beijing, Guizhou and Shandong provinces, China. In total, eighteen fungal strains that fitted well into the species concept of Diaporthe were isolated. Based on both morphological and multi-locus phylogenetic analyses of internal transcribed spacer region (ITS), beta-tubulin (tub-2), calmodulin (Cal) and translation elongation factor 1-α (tef1-α) sequencing data, fourteen isolates were identified as Diaporthe eres, while four isolates were classified as D. hongkongensis. Here, we report D. hongkongensis causing sweet cherry branch dieback disease and, further, we confirmed the host association of D. eres with sweet cherries in China. A pathogenicity assay revealed the ability of both D. eres and D. hongkongensis to cause shoot necrosis and stem lesions on Prunus avium cv. 'Brooks' (mean lesion lengths of 1.86 cm and 1.56 cm, respectively). The optimal temperature for the growth of both Diaporthe species was tested. The optimal growth temperature for D. hongkongensis was 30 °C, and the 25-28 °C temperatures were the most favorable for the growth of D. eres strains. This research advances the understanding of fungal trunk diseases in fruit crops, particularly gummosis and branch dieback disease in Chinese cherry orchards, and will aid growers in making decisions about cultural practices and disease management.

Development of PCR based SSR markers for Wilsonomyces carpophilus and a PCR based diagnosis protocol for the early detection of shot hole disease in stone fruit crops.

BACKGROUND: The conidial Ascomycota fungus Wilsonomyces carpophilus causing shot hole in stone fruits is a major constraint in the production of stone fruits worldwide. Shothole disease symptoms appear on leaves, fruits, and twigs. Successful isolation of the pathogen from different hosts on synthetic culture medium is a time consuming and tedious procedure for identification of the pathogen based on morpho-cultural characterization. METHODS AND RESULTS: The present research was carried out to develop a successful PCR based early detection protocol for the shot hole disease of stone fruits, viz., peach, plum, apricot, cherry, and almond using the pathogen specific SSR markers developed from the Wilsonomyces carpophilus genome using Genome-wide Microsatellite Analysing Tool package (GMATA) software. Diseased leaf samples of different stone fruits were collected from the SKUAST-K orchard and the pathogen was isolated on potato dextrose agar (PDA) medium and maintained on Asthana and Hawkers' medium with a total of 50 pathogen isolates comprised of 10 isolates each from peach, plum, apricot, cherry and almond. The DNA was extracted from both healthy and infected leaf samples of different stone fruits. The DNA was also extracted from the isolated pathogen cultures (50 isolates). Out of 2851 SSR markers developed, 30 SSRs were used for the successful amplification of DNA extracted from all the 50 pathogen isolates. These SSRs were used for the amplification DNA from shot hole infected leaf samples of different stone fruits, but the amplification was not observed in the control samples (DNA from healthy leaves), thus confirming the detection of this disease directly from the shot hole infected samples using PCR based SSR markers. To our knowledge, this forms the first report of SSR development for the Wilsonomyces carpophilus and their validation for the detection of shot hole disease directly from infected leaves. CONCLUSION: PCR based SSR makers were successfully developed and used for the detection of Wilsonomyces carpophilus causing shot hole disease in stone fruits including almond in nuts for the first time. These SSR markers could successfully detect the pathogen directly from the infected leaves of stone fruits namely peach, plum, apricot and cherry including almond from the nuts.

Estimation of Soluble Solids for Stone Fruit Varieties Based on Near-Infrared Spectra Using Machine Learning Techniques.

The quality control for fruit maturity inspection is a key issue in fruit packaging and international trade. The quantification of Soluble Solids (SS) in fruits gives a good approximation of the total sugar concentration at the ripe stage, and on the other hand, SS alone or in combination with acidity is highly related to the acceptability of the fruit by consumers. The non-destructive analysis based on Visible (VIS) and Near-Infrared (NIR) spectroscopy has become a popular technique for the assessment of fruit quality. To improve the accuracy of fruit maturity inspection, VIS−NIR spectra models based on machine learning techniques are proposed for the non-destructive evaluation of soluble solids in considering a range of variations associated with varieties of stones fruit species (peach, nectarine, and plum). In this work, we propose a novel approach based on a Convolutional Neural Network (CNN) for the classification of the fruits into species and then a Feedforward Neural Network (FNN) to extract the information of VIS−NIR spectra to estimate the SS content of the fruit associated to several varieties. A classification accuracy of 98.9% was obtained for the CNN classification model and a correlation coefficient of Rc>0.7109 for the SS estimation of the FNN models was obtained. The results reported show the potential of this method for a fast and on-line classification of fruits and estimation of SS concentration.

Melatonin triggers tissue-specific changes in anthocyanin and hormonal contents during postharvest decay of Angeleno plums.

Plum is a stone fruit that stands out for having a short shelf-life because of its high susceptibility to rapid deterioration. Part of this deterioration is explained by fruit overripening. Recently, the role of melatonin in delaying postharvest decay has been investigated but its regulatory function during overripening is still under extensive debate. In this study, to understand physiological events taking place in plums overripening and elucidate the role of melatonin on the postharvest quality of these fruits and its relationship to other plant hormones, Angeleno plums were sprayed with 10-4 M of melatonin solution immediately after harvest. We carried out tissue-specific (mesocarp and exocarp) analysis of total phenols and anthocyanin quantification, as well as the evaluation of different phytohormones by LC-MS/MS and fruit quality parameters. Results showed that during postharvest, endogenous melatonin contents decreased both in the mesocarp and the exocarp of Angeleno plums. Likewise, plum firmness also decreased and a strong correlation was found for this parameter with jasmonic acid (JA) and cytokinins. Conversely, after exogenous melatonin application, endogenous melatonin content increased both in mesocarp and exocarp but it had a differential effect depending on the plum tissue. Indeed, total phenol and anthocyanin contents arose by 21% and 58%, respectively, in the mesocarp after melatonin treatment but no variations were found in the exocarp of Angeleno plums. Hormonal analysis of Angeleno mesocarp also revealed an increase in the JA and its precursor, 12-oxo-phytodienoic acid (OPDA), on the fourth day after melatonin application as well as a positive correlation between melatonin and gibberellin 1 (GA1). These results suggest that melatonin may be acting as a signal molecule increasing phenolic compounds contents through direct regulation and by signaling with other phytohormones. Therefore, this research provides valuable information for understanding the regulatory role of melatonin and its relationship with plant hormones during overripening to contribute to improve the postharvest quality of plums.

QTLs Identification for Iron Chlorosis in a Segregating Peach-Almond Progeny Through Double-Digest Sequence-Based Genotyping (SBG).

Linkage maps are highly appreciated tools for cultivar and rootstock breeding programs because they are suitable for genetic and genomic studies. In this study, we report on using sequence-based genotyping (SBG) approach to simultaneously discover and genotype SNPs from two peach-based rootstocks ("Adafuel" and "Flordaguard") and their progeny (n = 118): from a initial mapping population composed of 131 seedlings. The plant material was developed at the EEAD-CSIC Prunus rootstocks breeding program, aiming to obtain a segregating progeny for a range of characters of agronomical interest to rootstock breeding (iron-chlorosis and root-asphyxia tolerance, nematode resistance, vigor traits, and other effects on scion cultivars). Sequence reads obtained from double-digest SBG were aligned to the P. persica reference genome (Peach v2.0). While eight linkage groups were constructed for "Adafuel," only four linkage groups were constructed for "Flordaguard," given the low heterozygosity of this last genotype. High synteny and co-linearity were observed between obtained maps and Peach v2.0. On the other hand, this work aimed to elucidate the genetic basis of leaf chlorosis tolerance using the phenotypic segregation of the progeny to iron-chlorosis tolerance, along with the QTLs responsible for leaf chlorosis. The F1 mapping population, composed initially of 131 seedlings, was growing in four field trials established on calcareous soils at the experimental field of the EEAD-CSIC in Zaragoza, Spain. From the initial mapping population, 131 individuals were selected for their phenotypical characterization with SPAD measurements of plants grown in the field, exhibiting a great variability. Significant QTLs associated with tolerance to iron chlorosis were found in LG1, LG5, LG7, and LG8. The significant QTLs detected in LG5 and LG7 have not been associated with this abiotic stress before in Prunus. Several candidate genes such as Prupe.1G541100, predicted as glutamyl-tRNA reductase 1, Prupe.1G468200, encoding a 2-oxoglutarate (2OG), and Fe(II)-dependent oxygenase superfamily protein or Prupe.1G577000 (ppa011050.m), a NIFU-like protein 2 (NIFU2) were detected. The exact biological function of some of these genes should be verified for the future development of marker-assisted selection for peach iron chlorosis tolerance.

Application of Glycine betaine coated chitosan nanoparticles alleviate chilling injury and maintain quality of plum (Prunus domestica L.) fruit.

The use of edible coatings can lead to significant extension of the postharvest life of fresh horticultural products through the regulation of water and gaseous exchange during storage. In this regard, nano-engineered materials are of great interest to design novel and multifunctional edible coatings and are increasingly employed. Chitosan and glycine betaine have been reported to enhance fruit tolerance to chilling stress during cold storage. The current study applied new coating treatments to plum (Prunus domestica L. cv. 'Stanley') fruit at maturity stage in a completely randomized factorial design with three replicates. Plums were treated with distilled water (control), glycine betaine (GB) at 2.5 and 5 mM, chitosan (CTS) at 1% (w/v) or glycine betaine-coated chitosan nanoparticles (CTS-GB NPs) at 0.5 and 1% (w/v) and stored at 1 °C for up to 40 days. The application of CTS-GB NPs (0.5% w/v) was the most effective treatment and induced lower electrolyte leakage, MDA and H2O2 content, and significantly alleviated chilling injury. Furthermore, this treatment remarkably increased the activity of PAL enzyme, resulting in higher levels of phenolics, flavonoids and anthocyanins content, and enhanced DPPH scavenging capacity. In addition, CTS-GB NPs treatment increased endogenous GB (9.25 mg g-1 DW) and proline (1929.29 µg g-1 FW) accumulation leading to higher activity of CAT, POD, SOD and APX enzymes. Based on the obtained results, the commercial application of CTS-GB NPs could effectively reduce chilling injury, preserve nutritional quality, and prolong the storage potential and shelf life of plum fruit.

Oxytetracycline and Streptomycin Resistance Genes in Xanthomonas arboricola pv. pruni, the Causal Agent of Bacterial Spot in Peach.

Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot, a major worldwide disease of Prunus species. Very few chemical management options are available for this disease and frequent applications of oxytetracycline (OTC) in the United States peach orchards have raised concerns about resistance development. During 2017-2020, 430 Xap strains were collected from ten peach orchards in South Carolina. Seven OTC-resistant (OTC R ) Xap strains were found in 2017 and 2020 from four orchards about 20-270 km apart. Interestingly, the seven strains were also resistant to streptomycin (STR). Six strains grew on media amended with ≤100 µg/mL OTC, while one strain, R1, grew on ≤250 µg/mL OTC. Genome sequence analysis of four representative OTC R strains revealed a 14-20 kb plasmid carrying tetC, tetR, and strAB in each strain. These three genes were transferable to Xanthomonas perforans via conjugation, and they were PCR confirmed in all seven OTC R Xap strains. When tetC and tetR were cloned and expressed together in a sensitive strain, the transconjugants showed resistance to ≤100 µg/mL OTC. When tetC was cloned and expressed alone in a sensitive strain, the transconjugants showed resistance to ≤250 µg/mL OTC. TetC and tetR expression was inducible by OTC in all six wild-type strains resistant to ≤100 µg/mL OTC. However, in the R1 strain resistant to ≤250 µg/mL OTC, tetR was not expressed, possibly due to the presence of Tn3 in the tetR gene, and in this case tetC was constitutively expressed. These data suggest that tetC confers OTC resistance in Xap strains, and tetR regulates the level of OTC resistance conferred by tetC. To our knowledge, this is the first report of OTC resistance in plant pathogenic xanthomonads.

Taxonomic Refinement of Xanthomonas arboricola.

Xanthomonas arboricola comprises a number of economically important fruit tree pathogens classified within different pathovars. Dozens of nonpathogenic and taxonomically unvalidated strains are also designated as X. arboricola, leading to a complicated taxonomic status in the species. In this study, we have evaluated the whole-genome resources of all available Xanthomonas spp. strains designated as X. arboricola in the public databases to refine the members of the species based on DNA similarity indexes and core genome-based phylogeny. Our results show that, of the nine validly described pathovars within X. arboricola, pathotype strains of seven pathovars are taxonomically genuine, belonging to the core clade of the species regardless of their pathogenicity on the host of isolation (thus the validity of pathovar status). However, strains of X. arboricola pv. guizotiae and X. arboricola pv. populi do not belong to X. arboricola because of the low DNA similarities between the type strain of the species and the pathotype strains of these two pathovars. Thus, we propose to elevate the two pathovars to the rank of a species as X. guizotiae sp. nov. with the type strain CFBP 7408T and X. populina sp. nov. with the type strain CFBP 3123T. In addition, other mislabeled strains of X. arboricola were scattered within Xanthomonas spp. that belong to previously described species or represent novel species that await formal description.

Modification of the existing maximum residue levels for fosetyl/phosphonic acid in apricots, cherries and plums resulting from the use of potassium phosphonates.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant LAINCO S.A. submitted a request to the competent national authority in Greece to modify the existing maximum residue levels (MRLs), currently expressed as fosetyl in the MRL Regulation, resulting from the use of the active substance potassium phosphonates on apricots, cherries and plums. The data submitted in support of the request were found to be sufficient to derive MRL proposals and address the data gap for residue trials compliant with the Southern EU uses identified in the joint MRL review for cherries and plums, but insufficient for apricots. Adequate analytical methods for enforcement are available to control the residues on the commodities under consideration. Based on the risk assessment results, EFSA concluded that the long-term intake of residues resulting from the uses of potassium phosphonates according to the intended agricultural practices is unlikely to present a risk to consumer health.

Listeria monocytogenes Survival on Peaches and Nectarines under Conditions Simulating Commercial Stone-Fruit Packinghouse Operations.

Recent recalls of stone fruit due to potential Listeria contamination and associated foodborne outbreaks highlight the risk for pathogen transmission through stone-fruit consumption. Particularly, surface contamination of fruits increases the risk for cross-contamination of produce during processing and storage. This highlights the need for quality control in stone fruits intended for consumption. To develop effective food safety practices, it is essential to determine the critical factors during stone-fruit processing that influence Listeria survival. Therefore, this study evaluated the ability of Listeria to survive on peaches and nectarines under simulated stone-fruit loading and staging, waxing and fungicide application and storage conditions. The results of our study indicate that current stone-fruit handling conditions do not favor Listeria growth. However, once fruit is contaminated, Listeria can survive on the fruit surface in significant numbers under current processing conditions. Therefore, there is a need to develop and implement preventive controls at the stone-fruit packinghouse to prevent Listeria contamination and deter pathogen persistence.

Climate-Aware and IoT-Enabled Selection of the Most Suitable Stone Fruit Tree Variety.

The application of new technologies such as the Internet of Things offers the opportunity to improve current agricultural development, facilitate daily tasks, and turn farms into efficient and sustainable production systems. The use of these new technologies enables the digital transformation process demanded by the sector and provides agricultural collectives with more optimized analysis and prediction tools. Due to climate change, one of the farm industry's problems is the advance or decay in the cycle of stone fruit trees. The objective is to recommend whether a specific area meets the minimum climatic requirements for planting certain stone fruit trees based on climatic data and bioclimatic indicators. The methodology used implements a large amount of meteorological data to generate information on specific climatic conditions and interactions on crops. In this work, a pilot study has been carried out in the Region of Murcia using an IoT platform. We simulate scenarios for the development of stone fruit varieties better adapted to the environment. Based on the standard, open interfaces, and protocols, the platform integrates heterogeneous information sources and interoperability with other third-party solutions to exchange and exploit such information.

Effect of Grafting on Viral Resistance of Non-transgenic Plum Scion Combined With Transgenic PPV-Resistant Rootstock.

In stone fruit trees, resistance to Plum pox virus (PPV) can be achieved through the specific degradation of viral RNA by the mechanism of RNA interference (RNAi). Transgenic virus-resistant plants, however, raise serious biosafety concerns due to the insertion and expression of hairpin constructs that usually contain various selective foreign genes. Since a mature stone tree represents a combination of scion and rootstock, grafting commercial varieties onto transgenic virus-tolerant rootstocks is a possible approach to mitigate biosafety problems. The present study was aimed at answering the following question: To what extent are molecular RNAi silencing signals transmitted across graft junctions in transgrafted plum trees and how much does it affect PPV resistance in genetically modified (GM)/non-transgenic (NT) counterparts? Two combinations, NT:GM and GM:NT (scion:rootstock), were studied, with an emphasis on the first transgrafting scenario. Viral inoculation was carried out on either the scion or the rootstock. The interspecific rootstock "Elita" [(Prunus pumila L. × P. salicina Lindl.) × (P. cerasifera Ehrh.)] was combined with cv. "Startovaya" (Prunus domestica L.) as a scion. Transgenic plum lines of both cultivars were transformed with a PPV-coat protein (CP)-derived intron-separate hairpin-RNA construct and displayed substantial viral resistance. High-throughput sequence data of small RNA (sRNA) pools indicated that the accumulation of construct-specific small interfering RNA (siRNA) in transgenic plum rootstock reached over 2%. The elevated siRNA level enabled the resistance to PPV and blocked the movement of the virus through the GM tissues into the NT partner when the transgenic tissues were inoculated. At the same time, the mobile siRNA signal was not moved from the GM rootstock to the target NT tissue to a level sufficient to trigger silencing of PPV transcripts and provide reliable viral resistance. The lack of mobility of transgene-derived siRNA molecules was accompanied by the transfer of various endogenous rootstock-specific sRNAs into the NT scion, indicating the exceptional transitivity failure of the studied RNAi signal. The results presented here indicate that transgrafting in woody fruit trees remains an unpredictable practice and needs further in-depth examination to deliver molecular silencing signals.

High-Throughput Screening and Characterization of Phenolic Compounds in Stone Fruits Waste by LC-ESI-QTOF-MS/MS and Their Potential Antioxidant Activities.

Stone fruits, including peach (Prunus persica L.), nectarine (Prunus nucipersica L.), plum (Prunus domestica L.) and apricot (Prunus armeniaca L.) are common commercial fruits in the market. However, a huge amount of stone fruits waste is produced throughout the food supply chain during picking, handling, processing, packaging, storage, transportation, retailing and final consumption. These stone fruits waste contain high phenolic content which are the main contributors to the antioxidant potential and associated health benefits. The antioxidant results showed that plum waste contained higher concentrations of total phenolic content (TPC) (0.94 ± 0.07 mg gallic acid equivalents (GAE)/g) and total flavonoid content (TFC) (0.34 ± 0.01 mg quercetin equivalents (QE)/g), while apricot waste contained a higher concentration of total tannin content (TTC) (0.19 ± 0.03 mg catechin equivalents (CE)/g) and DPPH activity (1.47 ± 0.12 mg ascorbic acid equivalents (AAE)/g). However, nectarine waste had higher antioxidant capacity in ferric reducing-antioxidant power (FRAP) (0.98 ± 0.02 mg AAE/g) and total antioxidant capacity (TAC) (0.91 ± 0.09 mg AAE/g) assays, while peach waste showed higher antioxidant capacity in 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay (0.43 ± 0.09 mg AAE/g) as compared to other stone fruits waste. Qualitative and quantitative phenolic analysis of Australian grown stone fruits waste were conducted by liquid chromatography coupled with electrospray-ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS) and HPLC-photodiode array detection (PDA). The LC-ESI-QTOF-MS/MS result indicates that 59 phenolic compounds were tentatively characterized in peach (33 compounds), nectarine (28), plum (38) and apricot (23). The HPLC-PDA indicated that p-hydroxybenzoic acid (18.64 ± 1.30 mg/g) was detected to be the most dominant phenolic acid and quercetin (19.68 ± 1.38 mg/g) was the most significant flavonoid in stone fruits waste. Hence, it could be concluded that stone fruit waste contains various phenolic compounds and have antioxidant potential. The results could support the applications of these stone fruit wastes in other food, feed, nutraceutical and pharmaceutical industries.

Genetic Transformation in Peach (Prunus persica L.): Challenges and Ways Forward.

Almost 30 years have passed since the first publication reporting regeneration of transformed peach plants. Nevertheless, the general applicability of genetic transformation of this species has not yet been established. Many strategies have been tested in order to obtain an efficient peach transformation system. Despite the amount of time and the efforts invested, the lack of success has significantly limited the utility of peach as a model genetic system for trees, despite its relatively short generation time; small, high-quality genome; and well-studied genetic resources. Additionally, the absence of efficient genetic transformation protocols precludes the application of many biotechnological tools in peach breeding programs. In this review, we provide an overview of research on regeneration and genetic transformation in this species and summarize novel strategies and procedures aimed at producing transgenic peaches. Promising future approaches to develop a robust peach transformation system are discussed, focusing on the main bottlenecks to success including the low efficiency of A. tumefaciens-mediated transformation, the low level of correspondence between cells competent for transformation and those that have regenerative competence, and the high rate of chimerism in the few shoots that are produced following transformation.

Aureobasidium pullulans volatile organic compounds as alternative postharvest method to control brown rot of stone fruits.

Volatile compounds produced by L1 and L8 strains were assayed against mycelia and conidia growth of Monilinia laxa, M. fructicola, M. polystroma, and M. fructigena of stone fruits. Results showed that volatile metabolites inhibited significantly pathogens growth, in particular M. fructigena mycelium growth (70% by L1 and 50% by L8) and M. fructicola conidia germination (85% by L1 and 70% by L8) compared to the control. Moreover, the antagonistic activity was enhanced by the addition of asparagine (120 mg L-1) in the culture media composition. Synthetic pure compounds were tested in vitro on pathogens mycelial and conidia growth and their EC50 values were estimated, confirming 2-phenethyl as the most active compound. For this reason 2-phenethyl and VOCs of both yeast strains were assayed in vivo on cherry, peach, and apricot fruits. Regarding peach fruit, both treatments, yeasts and pure compounds, displayed the best inhibiting action against all the pathogens especially against M. laxa (100% by L1, 84% by L8 and 2-phenethyl). ATR/IR spectroscopy analysis showed how VOCs produced by both strains increase the fruit waxes complexity reducing the pathogens attack so playing an essential role in the antagonistic activity of both yeast strains and on fruit structural composition.

Highly sensitive monoclonal antibody-based immunoassays for the analysis of fluopyram in food samples.

Monoclonal antibody-based techniques have become a useful analytical technology in the agro-food sector. Nowadays, residues of the recently registered fungicide fluopyram are increasingly being found in quality control programs. In the present study, novel chemical derivatives of this pesticide were prepared and specific and high-affinity monoclonal antibodies to fluopyram were raised for the first time. Moreover, immunoassays to fluopyram were developed in two alternative enzyme-linked immunosorbent assay formats, using homologous and heterologous assay conjugates, with limits of detection below 0.05 µg L-1. The optimized immunoassays were applied to the analysis of fluopyram in fortified plums and grapes of four different varieties as well as in in-house prepared musts and wines. Recoveries were between 76.3% and 109.6% and coefficients of variation were below 20%. Quantification limits were well below the maximum residue limits. Immunoassay performance was statistically validated with a reference chromatographic technique using samples from fluopyram-treated plum and grape cultivars.

Agrobacterium-Mediated Transformation of Russian Commercial Plum cv. "Startovaya" (Prunus domestica L.) With Virus-Derived Hairpin RNA Construct Confers Durable Resistance to PPV Infection in Mature Plants.

In modern horticulture Plum pox virus (PPV) imposes serious threats to commercial plantations of a wide range of fruit species belonging to genera Prunus. Given the lack of natural genetic resources, which display reliable resistance to PPV infection, there has been considerable interest in using genetic engineering methods for targeted genome modification of stone fruit trees to control Sharka disease caused by PPV. Among the many virus defense mechanisms, RNA interference is shown to be the most promising transgenic disease-control strategy in plant biotechnology. The present study describes the production of transgenic PPV resistant European plum "Startovaya" (P. domestica L.) through the Agrobacterium-mediated transformation of in vitro leaf explants. Due to organogenesis from leaves, the established protocol allows the genetic engineering of the plum genome without losing clonal fidelity of original cultivar. Seven independent transgenic plum lines containing the self-complementary fragments of PPV-CP gene sequence separated by a PDK intron were generated using hpt as a selective gene and uidA as a reporter gene. The transformation was verified through the histochemical staining for ß-glucuronidase activity, PCR amplification of appropriate vector products from isolated genomic DNA and Southern blot analysis of hairpin PPV-CP gene fragments. To clarify the virus resistance, plum buds infected by PPV-M strain were grafted onto 1-year-old transgenic plants, which further were grown into mature trees in the greenhouse. As evaluated by RT-PCR, DAS-ELISA, Western blot, ImmunoStrip test, and visual observations, GM plum trees remained uninfected over 9 years. Infected branches that developed from grafted buds displayed obvious symptoms of Sharka disease over the years and maintained the high level of virus accumulation, whereby host transgenic trees had been constantly challenged with the pathogen. Since the virus was unable to spread to transgenic tissues, the stable expression of PPV-derived gene construct encoding intron-spliced hairpin RNAs provided a highly effective protection of plum trees against permanent viral infection. At the same time, this observation indicates the lack of the systemic spread of resistance from GM tissues to an infected plum graft even after years of joint growth.

Association of a 'Candidatus Phytoplasma aurantifolia'-related strain with apricot showing European stone fruit yellows symptoms in Iran.

During 2012-2015 surveys in some orchards in Faraghe (Iran), a number of apricot trees showed symptoms resembling those associated with the phytoplasma disease known as European stone fruit yellows that are severe leaf roll, yellowing and die back. The presence of an infectious agent was confirmed by graft transmission experiments in which all the previously symptomless GF-677 (peach × almond) trees showed phytoplasma-type symptoms. The phytoplasma presence was confirmed by nested PCR assays using DNA extracted from samples from both field collected and graft-inoculated trees. The sequences of four nested PCR products from symptomatic apricot and experimentally graft-inoculated GF-677 trees were 100% identical to each other. RFLP and phylogenetic analyses carried out on these sequences allowed to cluster the strain infecting apricot trees in Iran with the16SrII-C subgroup phytoplasmas. This is the first report of a 16SrII-C phytoplasma associated with leaf roll and yellowing of the leaves in apricot trees.