First Report of Calosphaeria pulchella causing Canker and Twig Dieback of Peach (Prunus persica) in California, U.S.A.

California leads the United States in peach (Prunus persica L.) production, with approximately 505,000 tons produced in 2021 and valued at $378.3 million (California Agriculture Statistics Review, 2021-2022). During the spring and summer of 2023, twig and branch dieback were observed in three peach orchards (cvs. Late Ross and Starn) in San Joaquin County, California. Wood cankers and discoloration also occurred in branches, generally initiating at pruning wounds. Approximately 8 symptomatic twigs or branches per orchard were collected to proceed with the isolation of necrotic tissues on acidified potato dextrose agar (APDA). Isolations consistently yielded colonies of the fungal pathogen Calosphaeria pulchella (Pers. : Fr.) J. Schröt. (Réblová et al. 2004; Trouillas et al. 2012). Pure cultures were obtained by transferring single hyphal tips onto new APDA Petri plates. Colonies on APDA grew dark pink to red or purple in their center, with a white margin. Conidiogenesis was phialidic, producing round conidial masses at the tip of phialides. Conidia were produced abundantly on APDA, and were hyaline, allantoid to oblong-ellipsoidal, 4 to 5.5 (7) × 1.2 to 2.3 µm (n = 60). Two representative isolates (SJC-62 and SJC-64) were selected for genomic DNA extraction and sequencing of the internal transcribed spacer region (ITS) using ITS5/ITS4 universal primers and the beta-tubulin (TUB2) gene region using primers Bt2a and Bt2b. Consensus sequences of the two genes for the two isolates (ITS: PP063990, PP063991; TUB2: PP068303, PP068304) were compared to reference sequences (Réblová et al. 2015; Trouillas et al. 2012) using BLAST analysis. The ITS sequences of SJC-62 and SJC-64 were 99.8 and 99.5% identical to that of C. pulchella ex-type strain CBS 115999 (NR145357) and reference strain SS07 (HM237297); the TUB2 sequences were at least 98.5% identical to that of C. pulchella CBS 115999 (KT716476). Pathogenicity tests were conducted in 2- to 3-year-old healthy branches on 7-year-old peach trees, cvs. Loadel, Late Ross and Starn using the two fungal isolates and a control treatment (1 branch per treatment and 3 branches per tree) on each of 8-tree replicates. Branches were inoculated in June 2023 following wounding with a 5 mm cork borer to remove the bark and placing an agar plug from the margin of 10-day-old colonies on APDA directly into the fresh wound. Sterile agar plugs were used as controls. Inoculation sites were covered with petroleum jelly and wrapped with Parafilm to retain moisture. The experiment was completed twice. After four months, cankers and vascular discolorations developed around the inoculation sites. Length of vascular discoloration in inoculated branches averaged 72, 75, and 79 mm, for the Loadel, Starn, and Late Ross cvs., respectively. Calosphaeria pulchella was re-isolated from inoculated branches at 80 to 100% recovery rate, thus fulfilling Koch's postulates. The average length of vascular discoloration in the control was 13.5 mm and no fungi were recovered from control branches. Calosphaeria canker caused by C. pulchella is a global disease of sweet cherry. Recently, it was reported to cause cankers in peach trees in Chile (Grinbergs et al. 2023). To our knowledge, this is the first report of C. pulchella causing cankers and twig dieback of peach trees in the United States. These findings improve our knowledge of the etiology of canker diseases affecting peach trees and is critical for the development of effective disease management strategies.

The Expanding Menagerie of Prunus-Infecting Luteoviruses.

Members of the genus Luteovirus are responsible for economically destructive plant diseases worldwide. Over the past few years, three luteoviruses infecting Prunus trees have been characterized. However, the biological properties, prevalence, and genetic diversity of those viruses have not yet been studied. High-throughput sequencing of samples of various wild, cultivated, and ornamental Prunus species enabled the identification of four novel species in the genus Luteovirus for which we obtained complete or nearly complete genomes. Additionally, we identified another new putative species recovered from Sequence Read Archive data. Furthermore, we conducted a survey on peach-infecting luteoviruses in eight European countries. Analyses of 350 leaf samples collected from germplasm, production orchards, and private gardens showed that peach-associated luteovirus (PaLV), nectarine stem pitting-associated virus (NSPaV), and a novel luteovirus, peach-associated luteovirus 2 (PaLV2), are present in all countries; the most prevalent virus was NSPaV, followed by PaLV. The genetic diversity of these viruses was also analyzed. Moreover, the biological indexing on GF305 peach indicator plants demonstrated that PaLV and PaLV2, like NSPaV, are transmitted by graft at relatively low rates. No clear viral symptoms have been observed in either graft-inoculated GF305 indicators or different peach tree varieties observed in an orchard. The data generated during this study provide a broader overview of the genetic diversity, geographical distribution, and prevalence of peach-infecting luteoviruses and suggest that these viruses are likely asymptomatic in peach under most circumstances.

Tranzschelia discolor as the Causal Agent of Rust on Nectarine, Peach, and Japanese Plum in Highland Areas of Northern Thailand.

A severe outbreak of rust disease was observed on Prunus species, P. persica (peach), P. persica var. nectarina (nectarine), and P. salicina (Japanese plum) cultivated in northern Thailand in the rainy season. Previous reports have identified the causal agents as Tranzschelia discolor and T. pruni-spinosae based on only morphological characteristics. Thus, the aim of this study was to identify rust fungi of Prunus spp. based on morphology and molecular analyses. Between May and July in 2020 and 2022, 18 isolates were collected from five areas in Chiang Mai and Chiang Rai provinces. Symptoms of rust consisted of cinnamon brown pustules of uredinia that were hypophyllous and visible as pale greenish to chlorotic yellowish angular spots on the upper leaf surfaces. Urediniospore shape, size, and color were similar to T. discolor. Molecular analysis of internal transcribed spacer (ITS) and partial 28S large subunit (LSU) region rRNA genes confirmed the isolates to be T. discolor. This is the first report of P. salicina as a host of T. discolor in Thailand.

First report of canker caused by Cytospora sorbicola on Sweet Cherry (Prunus avium) in Chile.

Chile leads cherry exports in the southern hemisphere with a total of 415.315 t exported in the 2022 to 2023 season (IQonsulting, 2023). Cytospora canker, produced by Cytospora spp., causes destructive infections and limit the productivity of sweet cherry orchards (Luo et al. 2019). This study was focused on isolating Cytospora strains to identify and characterize the species present in sweet cherry. During the period 2019-2022, ten samples of stem or branch presenting canker, dieback, gummosis or dead buds, were collected from sweet cherry cultivars 'Skeena', 'Lapins', 'Santina', 'Sweetheart', and 'Regina', in the regions Ñuble and O'Higgins, Chile. Five mm pieces from the necrotic wood margins of the samples were rinsed with sterile deionized water, placed on potato dextrose agar (PDA, Difco) and incubated at 20±2 ºC for 5 days. One isolate was recovered from each sample, resulting in ten Cytospora-like strains. Single hyphal tips were transferred onto PDA plates and all isolates were deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM). Colonies grown on PDA reached 89 mm in diameter in 10 d at 25 °C, showing irregular margin, lacking aerial mycelium, initially off-white to cream that turned greenish gray in the center, which darkens with age. After 20 days of culturing on pine needle agar (Chen et al. 2015), isolates produced conidiomata pycnidial, semi-immersed, black, and subglobose (362)445-555(681)×(357)528-700(1053) µm (n=10), generating amber slimy conidia masses; Conidiophores were phialidic, cylindrical, aseptate, hyaline (6.77)9-10.04(12.88)×(0.82)1.1-1.28(1.99) µm (n = 30); conidia were abundant, allantoid, hyaline to light brown, aseptate (3.39)4.28-4.57(5.36)×(0.69)0.96-1.09(1.47) µm (n = 30) (Supplementary Figure 1). No sexual morph was observed. With the exception of the strain RGM 3390, all the isolates shared morphological characters to the descriptions of Cytospora sorbicola Norphanph., Bulgakov, T. C. Wen & K. D. Hyde (Norphanphoun et al. 2017). Isolates were identified at species level, by sequencing DNA regions described by Pan et al. (2020): ITS1-5.8S-ITS2, LSU; act, tef-1α, and tub2 with the exception of the RBP2, because this region could not be amplified in seven out of ten isolates. The consensus tree included the concatenated sequences of the ten isolates and those of reference Cytospora species reported by Ilyukhin et al. (2023) using a maximum likelihood analysis with the tool IQ-TREE webserver. MLSA confirmed the taxonomic affiliation of nine of the isolates with C. sorbicola and one isolate with Cytospora sp. (RGM 3390), that might represent a novel species (Supplementary Figure 2). The isolates RGM 3399 and RGM 3400, were selected randomly for pathogenicity tests. Inoculations were performed on 2-year-old sweet cherry cv. 'Lapins' grow in pots in a greenhouse at 26±6°C. Seven plants per isolate were cut to about 6-cm length from the main stem, and inoculated onto fresh cuts with 5-mm mycelium PDA plugs of 5-d-old culture and wrapped in moist sterile cotton and parafilm to keep moisture. Six plants were inoculated with non-colonized PDA agar plugs as control. The average canker length 3 months after inoculation was 3.1 and 0.8 cm, for RGM 3389 and RGM 3400, respectively (Supplementary Figure 1). Symptomatic twigs were incubated in moist chambers at 20±2 ºC for 10 d, resulting in the re-isolation of Cytospora strains that produced pycnidia and conidia structures in agreement with C. sorbicola. Both strains were reidentified to fulfill Koch's postulates, control twigs remained asymptomatic and no fungus was isolated from these twigs. This is the first report of C. sorbicola causing canker on sweet cherry in Chile. Our findings suggest that this species could be the most recurrent in cherry in central Chile, coinciding with it found in California where C. sorbicola has been described as the main causal agent of Cytospora canker of stone fruits in California (Lawrence et al. 2018).

First Report of Brown Rot Caused by Monilinia polystroma on Sweet Cherry and Almond in Italy.

Brown rot decay is an important disease of pome and stone fruits. In Italy, the main pathogens on stone fruits are Monilinia laxa, M. fructigena, and M. fructicola (Spitaler et al. 2022a). In addition, Monilinia polystroma (G. Leeuwen) L. M. Kohn (van Leeuwen et al. 2002), was recently found in Italy on peach (Martini et al. 2014), pear (Martini et al. 2015), plum (Abate et al. 2018), apple (Rosati et al. 2021), and quince (Spitaler et al. 2022b). In South Tyrol province, sweet cherry (Prunus avium L.) and almond (Prunus dulcis Mill. D. A. Webb), plants of the Rosaceae family and belonging to stone fruits, were observed to be frequently affected by brown rot. Affected cherries as well as almonds showed brown lesions, covered by yellowish or buff-colored stroma in concentric rings. Symptomatic cherries became shriveled, while symptomatic almonds remained firm. To determine the pathogen, single spore isolates were obtained from five symptomatic fruits, each from a cherry orchard of the cultivar Kordia in July 2021 and almond trees of the variety Dulcis in August 2021. Both sample sites were situated in Vadena/Pfatten. Infestation in the cherry orchard, covered by a rain-protection foil, was determined to be about 1 %. In almond, over 50 % of the fruits of various ripening stages showed brown rot symptoms. On potato dextrose agar (PDA) at 22 °C and a photoperiod of 16 h, isolates from both fruits matched the morphological characteristics of M. polystroma (Vasic et al. 2016) within 14 days. DNA was extracted from mycelium and the rRNA encoding gene region using ITS4 and ITS6 primers as well as a genomic sequence of unknown function using the primers UniMon_Forw and UniMon_Rev (Petróczy et al. 2012), were amplified and sequenced. MegaBLAST analysis revealed 100 % identity with M. polystroma sequences of the NCBI GenBank (rRNA encoding region: NR_154198; genomic region: JN128836). Sequences were deposited in GenBank under the accession numbers OP642545/OP654171 (cherry) and OP642546/OP654172 (almond). Pathogenicity was confirmed with mature cherries cultivar Duroncino or almost mature almond fruit of the variety Dulcis, respectively: 16 samples each for both fruits were surface-sterilized by dipping in 75 % ethanol for 10 s and subsequent rinsing with sterile water for 10 s. Mycelial plugs (1 mm) were dislodged from a 7-day old colony and inserted in a 1 mm hole into the fruits. Incubation was performed in plastic boxes under the conditions described above. PDA-inoculated fruit were used as controls. All cherries and all almonds were completely covered by brown rot lesions 7 days and 15 days post inoculation, respectively. Control fruits remained symptomless. Conidia were produced in branched chains on mycelium-inoculated fruit. Conidia were one-celled, limoniform, hyaline, measuring 13.1 to 22.2 × 9.7 to 14.8 µm (cherry) and 14.1 to 20.8 × 10.7 to 15.3 µm (almond). Additionally, 16 fruits each were inoculated with 20 µL conidial suspension (5 x 10^3 spores/mL) from mycelium-inoculated fruits. All cherries as well as all almonds were completely covered by brown rot lesions 7 days and 15 days post inoculation. Control fruits remained symptomless. To confirm identity, the fungus was isolated from five spore-inoculated fruits each for cherry and almond. The isolates showed identical morphological features and sequence identity as the original isolates. To our knowledge, this is also the first report of M. polystroma on almond, while the pathogen has already been reported on sweet cherry in Poland (Poniatowska et al. 2016). These additional host plants identified in this study suggest a broad impact of M. polystroma on Italian stone fruit production. Due to the economically important cultivation of stone fruit, further knowledge about the pathogens' host range will help to assign brown rot symptoms to M. polystroma and to improve targeted control strategies.

Fungal trunk diseases causing decline of apricot and plum trees in the Czech Republic.

Fungal trunk diseases (FTDs) have been a significant threat to the global stone fruit industry. FTDs are caused by a consortium of wood-decaying fungi. These fungi colonize woody tissues, causing cankers, dieback, and other decline-related symptoms in host plants. In this study, a detailed screening of the fungal microbiota associated with the decline of stone fruit trees in the Czech Republic was performed. The wood fragments of plum and apricot trees showing symptoms of FTDs were subjected to fungal isolation. The partial internal transcribed spacer (ITS) region, partial beta-tubulin (tub2) and translation elongation factor 1-α (tef) genes were amplified from genomic DNA extracted from fungal cultures. All isolates were classified, and the taxonomic placement of pathogenic strains was illustrated in phylogenetic trees. The most abundant pathogenic genus was Dactylonectria (31 %), followed by Biscogniauxia (13 %), Thelonectria (10 %), Eutypa (9 %), Dothiorella (7 %), Diplodia (6 %), and Diaporthe (6 %). The most frequent endophytic genus was Aposphaeria (17 %). The pathogenicity of six fungal spp. (Cadophora daguensis, Collophorina africana, Cytospora sorbicola, Dothiorella sarmentorum, Eutypa lata, and Eutypa petrakii var. petrakii to four Prunus spp. was evaluated and the Koch's postulates were fulfilled. All tested isolates caused lesions on at least one Prunus sp. The most aggressive species was E. lata, which caused the largest lesions on all four tested Prunus spp., followed by E. petrakii var. petrakii, and D. sarmentorum. Japanese plum (Prunus salicina) and almond (P. amygdalus) were the most susceptible hosts while apricot (P. armeniaca) was the least susceptible host in the pathogenicity trial.

Resistance of Almond (Prunus dulcis) to Xylella fastidiosa: A Comparative Study on Cultivars.

Almond leaf scorch is a disease caused by Xylella fastidiosa, which is increasingly widespread globally in the main almond cultivation areas. Previously confined to America, in the last decade this disease has been reported in Iran and southern Europe. In this comparative study, the resistance to X. fastidiosa subsp. multiplex of 13 almond cultivars (Mamaei, Non-Pareil, Sefied, Rabie, Ferragnes, Shahrood21, Thompson, Merced, Marcona, Frudeul, Kapariel, Princess, and Tuono) grafted onto seedlings of Talkhe was evaluated in controlled conditions. Plants were artificially inoculated and maintained in greenhouse conditions. Approximately 3 months after inoculation, three times at 3-week intervals, disease incidence, disease severity, and disease index were determined based on scaling, and bacterial populations were estimated. The effect of winter survival of bacteria in outdoor potted seedlings was also investigated in all almond cultivars. Findings showed a great degree of variability in response to X. fastidiosa among cultivars considering symptom development and severity, as well as bacterial titer. Thompson and Rabie cultivars scored the best results from both a symptomatological and infectious point of view, indicating resistance against the pathogen compared with other tolerant cultivars (e.g., Ferragnes, Tuono, and Kapariel), thanks to the development of mild symptoms. Mamaei, Non-Pareil, and Sefied scored worst, suggesting a susceptible behavior when infected by X. fastidiosa. Given that the pathogen was not detected by culturing and PCR during the following summer, bacterial population in potted seedlings was reduced significantly by overwintering in outdoor conditions regardless of cultivar susceptibility. This suggests that cold treatment can be used as a preventive treatment to manage nursery almond seedlings.

Draft Genomic Resources for the Brown Rot Fungal Pathogen Monilinia laxa.

Monilinia laxa is the causal agent of brown rot on stone fruit, and it can cause heavy yield losses during field production and postharvest storage. This article reports the draft genome assembly of the M. laxa Mlax316 strain, obtained using a hybrid genome assembly with both Illumina short-reads and PacBio long-reads sequencing technologies. The complete draft genome consists of 49 scaffolds with total size of 42.81 Mb, and scaffold N50 of 2,449.4 kb. Annotation of the M. laxa assembly identified 11,163 genes and 12,424 proteins which were functionally annotated. This new genome draft improves current genomic resources available for M. laxa and represents a useful tool for further research into its interactions with host plants and into evolution in the Monilinia genus.

Phloem Metabolites of Prunus Sp. Rather than Infection with Candidatus Phytoplasma Prunorum Influence Feeding Behavior of Cacopsylla pruni Nymphs.

Phytoplasmas are specialized small bacteria restricted to the phloem tissue and spread by hemipterans feeding on plant sieve tube elements. As for many other plant pathogens, it is known that phytoplasmas alter the chemistry of their hosts. Most research on phytoplasma-plant interactions focused on the induction of plant volatiles and phytohormones. Little is known about the influence of phytoplasma infections on the nutritional composition of phloem and consequences on vector behavior and development. The plum psyllid Cacopsylla pruni transmits 'Candidatus Phytoplasma prunorum', the causing agent of European Stone Fruit Yellows (ESFY). While several Prunus species are susceptible for psyllid feeding, they show different responses to the pathogen. We studied the possible modulation of plant-insect interactions by bacteria-induced changes in phloem sap chemistry. Therefore, we sampled phloem sap from phytoplasma-infected and non-infected Prunus persica and Prunus insititia plants, which differ in their susceptibility to ESFY and psyllid feeding. Furthermore, the feeding behavior and development of C. pruni nymphs was compared on infected and non-infected P. persica and P. insititia plants. Phytoplasma infection did not affect phloem consumption by C. pruni nymphs nor their development time. In contrast, the study revealed significant differences between P. insititia and P. persica in terms of both phloem chemistry and feeding behavior of C. pruni nymphs. Phloem feeding phases were four times longer on P. insititia than on P. persica, resulting in a decreased development time and higher mortality of vector insects on P. persica plants. These findings explain the low infestation rates of peach cultivars with plum psyllids commonly found in field surveys.

Identification of Pseudomonas Isolates Associated With Bacterial Canker of Stone Fruit Trees in the Western Cape, South Africa.

Bacterial canker is a common bacterial disease of stone fruit trees. The causal agents responsible for the disease include several pathovars in Pseudomonas syringae sensu lato and newly described Pseudomonas species. Pseudomonad strains were isolated from symptomatic stone fruit trees, namely apricot, peach, and plum trees cultivated in spatially separated orchards in the Western Cape. A polyphasic approach was used to identify and characterize these strains. Using a multilocus sequence typing approach of four housekeeping loci, namely cts, gapA, gyrB, and rpoD, the pseudomonad strains were delineated into two phylogenetic groups within P. syringae sensu lato: P. syringae sensu stricto and Pseudomonas viridiflava. These results were further supported by LOPAT diagnostic assays and analysis of clades in the rep-PCR dendrogram. The pseudomonad strains were pathogenic on both apricot and plum seedlings, indicative of a lack of host specificity between Pseudomonas strains infecting Prunus spp. This is a first report of P. viridiflava isolated from plum trees showing symptoms of bacterial canker. P. viridiflava is considered to be an opportunistic pathogen that causes foliar diseases of vegetable crops, fruit trees, and aromatic herbs, and thus the isolation of pathogenic P. viridiflava from twigs of plum trees showing symptoms of bacterial canker suggests that this bacterial species is a potentially emerging stem canker pathogen of stone fruit trees in South Africa.

Evaluation of the Intelligent Sprayer System in Peach Production.

Air-blast sprayers are routinely used to apply pesticides in commercial peach orchards, with growers using both conventional air-blast and ultrasonic sensor-driven models. Even with advanced spray technologies, there are still concerns with the amount of chemicals used and lost to drift. Our study evaluated a laser sensor-based variable-rate sprayer system in three experimental peach orchards for pest and brown rot disease control, spray volume output, spray coverage, and spray drift. A single 378-liter air-blast sprayer was used for both the conventional air-blast and the Intelligent Sprayer (iSprayer) treatments. Treatments were started at the phenological stage of bloom and continued through final swell. The iSprayer treatment was as effective in controlling pests and brown rot disease as the conventional air-blast treatment. Compared with the conventional air-blast treatment, the iSprayer treatment reduced the spray volume (liters/hectare) in cultivar PF23 by 71% at bloom, 62% at pit hardening, and 55% at final swell. For Juneprince, the spray volume reduction was 50% at bloom, 40% at pit hardening, and 13% at final swell. Spray drift was significantly (P < 0.05) reduced only at bloom in the iSprayer treatment. Spray coverage was increased by 50.13 and 26.67% in the iSprayer treatment at bloom and pit hardening, respectively, but not at final swell. Our results show that the iSprayer maintained pest and disease control efficacy in peach orchards while reducing spray volume and drift compared with the conventional air-blast treatment.

High-Throughput Sequencing Analysis of the Bacterial Community in Stone Fruit Phloem Tissues Infected by "Candidatus Phytoplasma prunorum".

"Candidatus Phytoplasma prunorum" (CPp) is a highly destructive phytopathogenic agent in many stone fruit-growing regions in Europe and the surrounding countries. In this work, we focused on documenting entire bacterial community in the phloem tissues of 60 stone fruit trees. Nested PCR and two real-time PCR assays were used to select CPp-positive (group A) and CPp-negative samples (group B). Afterwards, high-throughput amplicon sequencing was performed to assess bacterial community compositions in phloem tissues. The bacterial composition in phloem tissue consisted of 118 distinct genera, represented mainly by Pseudomonas, Acinetobacter, Methylobacterium, Sphingomonas, and Rhizobium. Statistics showed that CPp influenced the bacterial composition of infected plants (group A) and that the bacterial community depended on the geographical origin of the sample. This is the first work focusing on an analysis of the influence of CPp on the bacteria coexisting in the phloem tissues of stone fruit trees.

Impact of Postharvest Storage on the Infection and Colonization of Penicillium digitatum and Penicillium expansum on Nectarine.

Very few studies have investigated the host-pathogen interaction of Penicillium spp. on nectarine. Penicillium digitatum was identified as pathogenic and highly aggressive on nectarine. A strong association was made with host age/ripeness. This points to a new mechanism or life strategy used by P. digitatum to infect and colonize previously thought nonhosts. The aim of this study was to determine the effect of postharvest storage of nectarine on the infection and colonization of P. digitatum and Penicillium expansum at molecular and physical (firmness and pH) levels. The impact of environmental conditions (cold storage) and pathogen pressure (inoculum load) was also investigated. Although disease incidence was much lower, lesions caused by P. digitatum were similar in size to those caused by P. expansum on freshly harvested nectarine. Disease incidence and lesion diameter significantly increased (larger than P. expansum) on longer stored fruit. Cold storage had the largest effect on P. digitatum. Inoculum load had a meaningful effect on both Penicillium spp. Storage significantly affected pH modulation and gene expression. The pathogens not only decreased but also, increased and maintained (similar to initial pH of the host) pH of infected tissue. The polygalacturonase (PG) gene and creA were upregulated by P. digitatum on 7-day postharvest fruit (other genes were unaffected). It partly explains the larger lesions on older or riper fruit. A different expression profile was observed from P. expansum: strong downregulation in PG and slight upregulation in pacC. Very different life strategies were used by the two Penicillium spp. when infecting nectarine. Unlike what is known on citrus, P. digitatum showed an opportunistic lifestyle that takes advantage of specific host and environmental conditions. It is largely still unclear (gene expression) what specifically triggers the increase in disease incidence (infection) and lesion diameter (colonization) of P. digitatum on older or riper fruit. The differences between in vivo and in vitro studies make it difficult to directly correlate results. Additional research is still needed to differentiate and understand the infection and colonization of these pathogens on the same host.

Preventive Fungicidal Control of Cytospora leucostoma in Peach Orchards in Colorado.

In western Colorado, Cytospora leucostoma is ubiquitous in peach orchards and has developed into a major limiting factor of peach production. The pathogen is unable to invade healthy intact phloem tissue of the tree, but instead, it requires a wound as a mode of entry. Bark injuries caused by cold and pruning in commercial orchard systems provide infection courts that, in suitable environment conditions, can lead to many successful fungal infections. Preventive fungicide control is an integral component of management in tree fruit production. Eighteen fungicides were tested at selected label dose rates for C. leucostoma control. All treatments were initially tested in vitro in fungicide-amended media dishes. Successful treatments were then tested under controlled conditions on detached peach branch segments. Effective fungicides identified in the laboratory assays (thiophanate-methyl, captan, lime sulfur, and copper hydroxide) were further tested as spray applications in the field and as wound sealant applications in combination with latex paint and kaolin clay. Of the treatments evaluated, thiophanate-methyl, captan, 50% latex paint, thiophanate-methyl amended in 50% latex paint, captan amended in 50% latex paint, and lime sulfur were most effective in reducing C. leucostoma necrotic area. Copper hydroxide was ineffective in all field trials and in some instances, yielded larger necrotic areas than the nontreated positive control shoots.

Production, pomological and nutraceutical properties of apricot.

Apricot (Prunus sp.) is an important fruit crop worldwide. Despite recent advances in apricot research, much is still to be done to improve its productivity and environmental adaptability. The availability of wild apricot germplasms with economically interesting traits is a strong incentive to increase research panels toward improving its economic, environmental and nutritional characteristics. New technologies and genomic studies have generated a large amount of raw data that the mining and exploitation can help decrypt the biology of apricot and enhance its agronomic values. Here, we outline recent findings in relation to apricot production, pomological and nutraceutical properties. In particular, we retrace its origin from central Asia and the path it took to attain Europe and other production areas around the Mediterranean basin while locating it in the rosaceae family and referring to its genetic diversities and new attempts of classification. The production, nutritional, and nutraceutical importance of apricot are recapped in an easy readable and comparable way. We also highlight and discuss the effects of late frost damages on apricot production over different growth stages, from swollen buds to green fruits formation. Issues related to the length of production season and biotic and abiotic environmental challenges are also discussed with future perspective on how to lengthen the production season without compromising the fruit quality and productivity.

De novo assembly and comparative transcriptome analysis of Monilinia fructicola, Monilinia laxa and Monilinia fructigena, the causal agents of brown rot on stone fruits.

BACKGROUND: Brown rots are important fungal diseases of stone and pome fruits. They are caused by several Monilinia species but M. fructicola, M. laxa and M. fructigena are the most common all over the world. Although they have been intensively studied, the availability of genomic and transcriptomic data in public databases is still scant. We sequenced, assembled and annotated the transcriptomes of the three pathogens using mRNA from germinating conidia and actively growing mycelia of two isolates of opposite mating types per each species for comparative transcriptome analyses. RESULTS: Illumina sequencing was used to generate about 70 million of paired-end reads per species, that were de novo assembled in 33,861 contigs for M. fructicola, 31,103 for M. laxa and 28,890 for M. fructigena. Approximately, 50% of the assembled contigs had significant hits when blasted against the NCBI non-redundant protein database and top-hits results were represented by Botrytis cinerea, Sclerotinia sclerotiorum and Sclerotinia borealis proteins. More than 90% of the obtained sequences were complete, the percentage of duplications was always less than 14% and fragmented and missing transcripts less than 5%. Orthologous transcripts were identified by tBLASTn analysis using the B. cinerea proteome as reference. Comparative transcriptome analyses revealed 65 transcripts over-expressed (FC ≥ 8 and FDR ≤ 0.05) or unique in M. fructicola, 30 in M. laxa and 31 in M. fructigena. Transcripts were involved in processes affecting fungal development, diversity and host-pathogen interactions, such as plant cell wall-degrading and detoxifying enzymes, zinc finger transcription factors, MFS transporters, cell surface proteins, key enzymes in biosynthesis and metabolism of antibiotics and toxins, and transposable elements. CONCLUSIONS: This is the first large-scale reconstruction and annotation of the complete transcriptomes of M. fructicola, M. laxa and M. fructigena and the first comparative transcriptome analysis among the three pathogens revealing differentially expressed genes with potential important roles in metabolic and physiological processes related to fungal morphogenesis and development, diversity and pathogenesis which need further investigations. We believe that the data obtained represent a cornerstone for research aimed at improving knowledge on the population biology, physiology and plant-pathogen interactions of these important phytopathogenic fungi.

Current achievements and future directions in genetic engineering of European plum (Prunus domestica L.).

In most woody fruit species, transformation and regeneration are difficult. However, European plum (Prunus domestica) has been shown to be amenable to genetic improvement technologies from classical hybridization, to genetic engineering, to rapid cycle crop breeding ('FasTrack' breeding). Since the first report on European plum transformation with marker genes in the early 90 s, numerous manuscripts have been published reporting the generation of new clones with agronomically interesting traits, such as pests, diseases and/or abiotic stress resistance, shorter juvenile period, dwarfing, continuous flowering, etc. This review focuses on the main advances in genetic transformation of European plum achieved to date, and the lines of work that are converting genetic engineering into a contemporary breeding tool for this species.

Influence of temperature on decay, mycelium development and sporodochia production caused by Monilinia fructicola and M. laxa on stone fruits.

Brown rot on peaches and nectarines caused by Monilinia spp. results in significant economic losses in Europe. Experiments were conducted to study the effects of temperature (0-33 °C) on the temporal dynamics of decay and mycelium development and the subsequent sporulation on peaches and nectarine fruit infected by M. laxa and M. fructicola. The rates of decay and mycelium development increased with temperature from 0 °C to 25 °C for both Monilinia species. At 0 °C, decay was faster for M. laxa (0.20 cm2 days-1) than for M. fructicola (0.07 cm2 days-1); indeed, M. laxa was able to develop mycelia and sporodochia, but M. fructicola was not. At 4 and 20 °C, there were no differences in decay and mycelia development between the two Monilinia species. When temperature increased from 25 to 33 °C, the rates of fungal decay and mycelium development decreased. At 30 and 33 °C, M. fructicola decayed faster (0.94 and 1.2 cm2 days-1, respectively) than M. laxa (0.78 and 0.74 cm2 days-1, respectively) and could develop mycelia and produce sporodochia, whereas M. laxa failed at 33 °C. These results indicated that M. fructicola is better adapted to high temperatures, whereas M. laxa is better adapted to low temperatures. These results can be used to predict the relative importance of the two species during the season at a given site and to improve management strategies for brown rot in areas where both species are present.

Development of SCAR markers for rapid and specific detection of Pseudomonas syringae pv. morsprunorum races 1 and 2, using conventional and real-time PCR.

Specific primers were developed to detect the causal agent of stone fruit bacterial canker using conventional and real-time polymerase chain reaction (PCR) methods. PCR melting profile (PCR MP) used for analysis of diversity of Pseudomonas syringae strains, allowed to pinpoint the amplified fragments specific for P. syringae pv. morsprunorum race 1 (Psm1) and race 2 (Psm2), which were sequenced. Using obtained data, specific sequence characterised amplified region (SCAR) primers were designed. Conventional and real-time PCRs, using genomic DNA isolated from different bacterial strains belonging to the Pseudomonas genus, confirmed the specificity of selected primers. Additionally, the specificity of the selected DNA regions for Psm1 and Psm2 was confirmed by dot blot hybridisation. Conventional and real-time PCR assays enabled accurate detection of Psm1 and Psm2 in pure cultures and in plant material. For conventional PCR, the detection limits were the order of magnitude ~10(0) cfu/reaction for Psm1 and 10(1) cfu/reaction for Psm2 in pure cultures, while in plant material were 10(0)-10(1) cfu/reaction using primers for Psm1 and 3 × 10(2) cfu/reaction using primers for Psm2. Real-time PCR assays with SYBR Green I showed a higher limit of detection (LOD) - 10(0) cfu/reaction in both pure culture and in plant material for each primer pairs designed, which corresponds to 30-100 and 10-50 fg of DNA of Psm1 and Psm2, respectively. To our knowledge, this is the first PCR-based method for detection of the causal agents of bacterial canker of stone fruit trees.

Specific PCR and real-time PCR assays for detection and quantitation of 'Candidatus Phytoplasma phoenicium'.

Almond witches' broom (AlmWB) is a fast-spreading lethal disease of almond, peach and nectarine associated with 'Candidatus Phytoplasma phoenicium'. The development of PCR and quantitative real-time PCR (qPCR) assays for the sensitive and specific detection of the phytoplasma is of prime importance for early detection of 'Ca. P. phoenicium' and for epidemiological studies. The developed qPCR assay herein uses a TaqMan(®) probe labeled with Black Hole Quencher Plus. The specificity of the PCR and that of the qPCR detection protocols were tested on 17 phytoplasma isolates belonging to 11 phytoplasma 16S rRNA groups, on samples of almond, peach, nectarine, native plants and insects infected or uninfected with the phytoplasma. The developed assays showed high specificity against 'Ca. P. phoenicium' and no cross-reactivity against any other phytoplasma, plant or insect tested. The sensitivity of the developed PCR and qPCR assays was similar to the conventional nested PCR protocol using universal primers. The qPCR assay was further validated by quantitating AlmWB phytoplasma in different hosts, plant parts and potential insect vectors. The highest titers of 'Ca. P. phoenicium' were detected in the phloem tissues of stems and roots of almond and nectarine trees, where they averaged from 10(5) to 10(6) genomic units per nanogram of host DNA (GU/ng of DNA). The newly developed PCR and qPCR protocols are reliable, specific and sensitive methods that are easily applicable to high-throughput diagnosis of AlmWB in plants and insects and can be used for surveys of potential vectors and alternative hosts.