Pest categorisation of Diplodia bulgarica.

The EFSA Plant Health Panel performed a pest categorisation of Diplodia bulgarica, a clearly defined plant pathogenic fungus of the family Botryosphaeriaceae. The pathogen affects Malus domestica, M. sylvestris and Pyrus communis causing various symptoms such as canker, twig blight, gummosis, pre- and post-harvest fruit rot, dieback and tree decline. The pathogen is present in Asia (India, Iran, Türkiye) and in non-EU Europe (Serbia). Concerning the EU, the pathogen is present in Bulgaria and widespread in Germany. There is a key uncertainty on the geographical distribution of D. bulgarica worldwide and in the EU, because in the past, when molecular tools were not available, the pathogen might have been misidentified as other Diplodia species (e.g. D. intermedia, D. malorum, D. mutila, D. seriata) or other members of the Botryosphaeriaceae family affecting apple and pear based only on morphology and pathogenicity tests. Diplodia bulgarica is not included in Commission Implementing Regulation (EU) 2019/2072. Plants for planting, other than seeds, fresh fruits, and bark and wood of host plants as well as soil and other plant-growing media carrying plant debris are the main pathways for the further entry of the pathogen into the EU. Host availability and climate suitability factors are favourable for the further establishment of the pathogen in the EU. In the areas of its present distribution, including Germany, the pathogen has a direct impact on cultivated hosts. Phytosanitary measures are available to prevent the further introduction and spread of the pathogen into the EU. Diplodia bulgarica satisfies the criteria that are within the remit of EFSA to assess for this species to be regarded as potential Union quarantine pest.

Expression Analysis of AUX/IAA Family Genes in Apple Under Salt Stress.

Members of the auxin/indoleacetic acid (Aux/IAA) gene family in plants are primary auxin-responsive genes that play important roles in many aspects of plant development and in responses to abiotic stress. Recently, 33 Aux/IAA have been identified in the apple genome. The biological responses of MdIAAs to salt stress are still unknown. In this study, Malus zumi, Malus baccata, and Malus × domestica 'Fuji' plantlets were subjected to salt stress by supplementing hydroponic media with NaCl at various concentrations. M. zumi showed the strongest salt resistance, followed by 'Fuji', and M. baccata was the most sensitive to salt stress. Tissue-specific expression profiles of MdIAAs were determined by quantitative real-time polymerase chain reaction. When apple plantlets were subjected to salt stress, most of salt-responsive MdIAAs were up-regulated by 1 h, 3 h, and 6 h in roots, shoot tips, and leaves, respectively. Highly expressed MdIAAs in roots, especially for M. zumi, consisted with the salt tolerance of apple rootstocks. Transgenic apple calli were tolerant to salt stress when over-expressed salt-responsive genes, MdIAA8, -9, and -25. These results provide clues about salt resistance in these three Malus species, which helps apple breeding of salt tolerance by genetic transformation.

A Binary-Based Matrix Model for Malus Corolla Symmetry and Its Variational Significance.

Floral symmetry (corolla symmetry) has important biological significance in plant genetics and evolution. However, it is often multi-dimensional and difficult to quantify. Here, we constructed a multi-dimensional data matrix [X Y Z] by extracting three qualitative variables with binary properties (X: corolla regularity of interval and coplanarity; Y: petal regularity of shape and size; Z: petal local regularity of curling and wrinkle) from different dimensions of petals (overall to individual, and then to the local): all petals (corolla), individual petals, and local areas of petals. To quantitatively express the degree of Malus corolla symmetry, these variables were then combined with weight assignments (X: 22 > Y: 21 > Z: 20) based on their contributions to the corolla symmetry and the algorithm rule of converting binary to decimal values, which facilitated the unification of qualitative and quantitative analyses. Our results revealed significant reduction in degrees of Malus corolla symmetry along the direction of local to overall. Species showed higher degree of corolla symmetry than cultivars; however, taxa with stronger corolla symmetry might not necessarily be species. These findings provide new insights into the circumscription of Malus controversial species. The matrix model should be reference for future evaluation of angiosperm flower symmetry (lack of corolla fusion).

Serological and Molecular Detection of Latent Viruses in the Apple Germplasm Bank of Santa Catarina

Abstract The Apple Germplasm Bank (AGB) of Santa Catarina Agricultural Research and Rural Extension Company - Epagri, AGB-Epagri, is the largest of the genus Malus in Brazil. Twenty-eight main accessions of this bank were virus screened through DAS-ELISA, RT-PCR and IC-RT-PCR during two consecutive reproductive cycles, and each accession showed latent mixed infection by at least two species, among ASGV, ASPV and ACLSV. The combined use of diagnostic methods helped overcome inconsistencies commonly found in apple virus detection and was shown essential for the AGB-Epagri can be safely used as a source of genetic variability and for the exchange of virus-free propagative material.

Phenotypic variation of floral organs in Malus using frequency distribution functions.

BACKGROUND: Phenotypic diversity of floral organs plays an important role in plant systematic taxonomy and genetic variation studies. Previous research have focused on the direction of variation but disregarded its degree. Phenotypic variation (including directions and degrees) of 17 floral traits from wild to cultivated crabapples were explored by comparing their distributions and deviations in three different dimensions: floral organ number, size, and the shape. RESULTS: Except for petal number, petal length / petal width, and sepal length / sepal width, the analyzed floral traits of cultivated crabapples all showed downward distributed box bodies in box plot analysis and left deviations of fitted curves in frequency distribution function analysis when compared to the wild, which revealed consistent variation directions of petaloid conversion (pistils or stamens → petals), size miniaturization (large → small), and shape narrowness (petal shape: circular → elliptic; sepal shape: triangular → lanceolate). However, only seven floral traits exhibited significant differences in box plot analysis, while all of the traits in frequency distribution function analysis were obviously offset. The variation degrees were quantitatively characterized by sizing traits > shaping traits > numbering traits and by horizontal dimensions > radial dimensions. CONCLUSIONS: Frequency distribution function analysis was more sensitive than the box plot analysis, which constructed a theoretical basis for Malus flower type breeding and would provide a new quantitative method for future evaluation of floral variation among different groups of angiosperms at large.

Canker and Wood Rot Pathogens Present in Young Apple Trees and Propagation Material in the Western Cape of South Africa.

Canker and wood rot pathogens cause dieback and, in severe cases, the death of young apple trees. Recently, a higher occurrence of cankers was observed on 1-year-old apple trees in the Western Cape Province of South Africa. This study aimed to assess the phytosanitary status of nursery trees and propagation material as possible inoculum sources for canker pathogens. Thirteen 1-year-old apple orchards showing canker or dieback symptoms were sampled. Certified nursery apple trees were collected from four nurseries as well as scion and rootstock mother plant material. Isolations were made from the discoloration observed in the vascular tissue of the plant parts and from asymptomatic material. Possible canker and wood rot species were identified with PCR and sequence comparisons of the relevant gene regions and phylogenetic analyses. Similar canker and wood rot species were isolated from 1-year-old diseased apple trees, nursery apple trees, and the propagation material. Forty-five fungal species associated with canker or wood rot symptoms were identified. The top five most abundant fungal species found causing disease on commercial 1-year-old trees were also found in high numbers causing latent infection in certified apple nursery trees. These species were Didymosphaeria rubi-ulmifolii sensu lato, Diplodia seriata, Schizophyllum commune, Didymella pomorum, and Coniochaeta fasciculata, with D. rubi-ulmifolii sensu lato being the dominant species in both sampling materials. In all, 65% of certified nursery apple trees, 5% of scion shoots used for budding, and 21% of rooted rootstock cuttings from layer blocks had latent infections of canker and wood rot pathogens. Pathogenicity trials were conducted with isolates of 39 species, inoculated onto 2-year-old branches of 14-year-old Golden Delicious trees. All species caused lesions that were significantly longer than the control. This study confirmed the presence of canker and wood rot pathogens in apple propagation material as well as certified nursery apple trees, which will aid the improvement of management practices in nurseries.

Phenolic compounds present in fruit extracts of Malus spp. show antioxidative and pro-apoptotic effects on human gastric cancer cell lines.

The study aims to analyze the phenolic compounds in Malus spp. and evaluate their antioxidant and pro-apoptotic effects in BGC-803 gastric cancer cells. The results showed that cyanidin-3-galactoside was the main polyphenol in Malus "Royalty" (MR), while catechin, epicatechin, and proanthocyanidin B1 and B2 contents were higher in Malus "Cinnabar" (MC) and Malus micromalus (MM) than in MR fruits. The total polyphenol content, total flavonoid content, and antioxidative properties of Malus spp. fruits followed an order of MR > MC > MM. Fruit extracts could inhibit BGC-803 cells growth and induce apoptosis, with IC50 values of 0.47, 0.36, and 0.31 mg/ml for MR, MC, and MM, respectively. Furthermore, fruit extracts induced cell apoptosis through increasing Bcl-2 and Bcl-xl (pro-apoptosis) expression and inhibiting Bax and Bak (anti-apoptosis) expression, thereby accelerating cell apoptosis. Taken together, these results demonstrate that red fruits (i.e., MR and MC) were more effective against cancer cells than green fruits (i.e., MM). PRACTICAL APPLICATIONS: Fruits of Malus spp. are ≤5 cm in size, considerably smaller than apples. They are rich in various natural bioactive compounds that are often consumed as a dietary supplement or used as natural raw materials for function food. In the current study, it is comprehensively characterized profile and bioactivity of phenolic metabolites in Malus spp. fruits of different colors, and found that red fruits are more effective in reducing the free radicals and inducing cancer cells apoptosis than green fruits. These findings are valuable for food technologists and food manufacturers, especially those who produce crabapple supplement. The study investigated the molecular mechanism of how Malus spp. fruits exert anti-cancer functions. This lays a theoretical foundation for future research on developing anti-cancer function food and provides helpful guidance for fruit market management and fruit processing industry.

Valorization of Wild Apple (Malus spp.) By-Products as a Source of Essential Fatty Acids, Tocopherols and Phytosterols with Antimicrobial Activity.

The amplified production of fruit as well as burgeoning demand for plant-made food products have resulted in a sharp increase of waste. Currently, millions of tons of by-products are either being discarded or utilized rather ineffectively. However, these by-products may be processed and further incorporated as functional ingredients in making high-value food products with many physiological and biochemical effects. The chemical analysis of pomace oils using gas chromatography-mass spectrometry (GC/MS) and reversed-phase-liquid chromatography coupled with fluorescence detector (RP-HPLC/FLD) systems led to the identification and quantification of 56 individual lipophilic compounds including unsaturated, polyunsaturated and saturated fatty acids, as well as phytosterols and four homologs of tocopherol. The oils recovered from by-products of Malus spp. (particularly cv. "Ola") are rich in fatty acids such as linolenic (57.8%), α-linolenic (54.3%), and oleic (25.5%). The concentration of total tocopherols varied among the Malus species and dessert apples investigated, representing the range of 16.8⁻30.9 mg mL-1. The highest content of total tocopherols was found in M. Bernu prieks, followed by M. cv. "Ola", and M. × Soulardii pomace oils. A significantly higher amount of δ-tocopherol was established in the oil of M. Bernu prieks, indicating that this species could be utilized as a natural and cheap source of bioactive molecules. ß-Sitosterol was the prevalent compound determined in all tested pomace oils with a percentage distribution of 10.3⁻94.5%. The main triterpene identified in the oils was lupeol, which varied in the range of 0.1⁻66.3%. A targeted utilization of apple pomace would facilitate management of tons of by-products and benefit the environment and industry.

Process-based allometry describes the influence of management on orchard tree aboveground architecture.

We evaluated allometric relationships in length, diameter, and mass of branches for two variably managed orchard tree species (tart cherry, Prunus cerasus; apple, Malus spp.). The empirically estimated allometric exponents (a) of the orchard trees were described in the context of two processed-based allometry models that make predictions for a: the West, Brown and Enquist fractal branching model (WBE) and the recently introduced Flow Similarity model (FS). These allometric models make predictions about relationships in plant morphology (e.g., branch mass, diameter, length, volume, surface area) based on constraints imposed on plant growth by physical and physiological processes. We compared our empirical estimates of a to the model predictions to interpret the physiological implications of pruning and management in orchard systems. Our study found strong allometric relationships among the species and individuals studied with limited agreement with the expectations of either model. The 8/3-power law prediction of the mass ∼ diameter relationship by the WBE, indicative of biomechanical limitations, was marginally supported by this study. Length-including allometric relationships deviated from predictions of both models, but shift toward the expectation of flow similarity. In this way, managed orchard trees deviated from strict adherence to the idealized expectations of the models, but still fall within the range of model expectations in many cases despite intensive management.

Pest categorisation of Phyllosticta solitaria.

The Panel on Plant Health performed a pest categorisation of Phyllosticta solitaria, the causal agent of blotch of apple, for the EU. The pest is a well-defined fungal species and methods are available for its detection and identification. P. solitaria is present in Canada and the continental states of the USA. The pest is not known to occur in the EU and is listed in Annex IAI of Directive 2000/29/EC, meaning its introduction into the EU is prohibited. The major cultivated host is Malus domestica (apple), but wild Malus and Crataegus species may also be affected. All hosts and major pathways of entry of the pest into the EU are currently regulated. The disease is favoured by warm, wet weather during the growing season. Host availability and climate matching suggest that P. solitaria could establish in parts of the EU and further spread mainly by human-assisted means. The pest causes premature defoliation, fruit cracking and rot, and twig and branch cankers. At the beginning of the 20th century, disease incidences of 70-90% on fruit of untreated susceptible apple cultivars had been reported and the disease was considered as a limiting factor in the commercial production of those cultivars. Nowadays, the disease is rare in commercial apple orchards, probably due to regular fungicide sprays against other diseases. The pest introduction in the EU would potentially cause impacts to apple production. The main uncertainties concern the host range, the maximum distance of conidial dispersal by wind-driven rain, and the magnitude of potential impacts to the EU. P. solitaria meets all the criteria assessed by EFSA for consideration as a potential Union quarantine pest. The criteria for considering P. solitaria as a potential Union regulated non-quarantine pest are not met, since the pest is not known to occur in the EU.

Pest categorisation of small-spored Alternaria carrying the genes for the AM- or AK-toxin biosynthesis.

The Panel on Plant Health performed a pest categorisation of small-spored Alternaria carrying the genes for the AM- or AK-toxin biosynthesis, for the EU. The identity of the pests is clearly defined and reliable methods exist for their detection/identification. They are listed in Annex IIAI of Directive 2000/29/EC as Alternaria alternata (non-European pathogenic isolates). Their distribution in the EU is restricted though with some uncertainty. The AM-toxin producer Alternaria affect Malus spp. and Pyrus communis (European pear), whereas the AK-toxin producer affect Pyrus pyrifolia, Pyrus bretschneideri and Pyrus ussuriensis (Asian pears). The pests could potentially enter the EU on host-planting material and fruit originating in infested countries. There are no biotic/abiotic factors limiting their potential establishment and spread in the EU, as their epidemiology is similar to that of other well-established Alternaria spp. Apples and European pears are widespread in the EU; Japanese pears are also present, but no data was found on their abundance/distribution. In the infested areas, the pests cause premature defoliation, fruit spotting and rot resulting in yield/quality losses. It is expected that the introduction and spread of the pests in the EU could impact apple and pear production, although the magnitude is unknown. Cultural practices and chemical measures may reduce the inoculum and the disease, but they cannot eliminate the pests. Phytosanitary measures are available to mitigate the risk of introduction and spread of the pests. The pests do not meet all the criteria assessed by EFSA for consideration as potential Union quarantine pests, as they are not under official control in those EU restricted areas where they have been found. The pests do not meet all the criteria assessed by EFSA to consider them as Union regulated non-quarantine pests, as host plants for planting are not the main means of pest spread.

Interception of 'Candidatus Phytoplasma solani' in Brazil in apple tree propagative material imported from France / Interceptação de 'Candidatus Phytoplasma solani' no Brasil em material propagativo de macieira importado da França

Apple plants from France introduced in Brazil for research purposes were subjected to a phytosanitary analysis at the Plant Quarantine Laboratory of Embrapa Genetic Resources and Biotechnology (Cenargen). After grafting onto healthy apple rootstock, some plants showed phytoplasma-infection symptoms. Polymerase Chain Reaction (PCR) tests yielded DNA fragments of the expected size for phytoplasmas. DNA sequencing revealed an identity of the 16S rDNA nucleotide sequence of 98-99% with 'Candidatus Phytoplasma solani'. This phytoplasma species is responsible for losses in European apple orchards and has not been reported in Brazil. According to the Federal Legislation on Plant Protection, the plants were incinerated to avoid the introduction of this exotic pest in Brazil.(AU)

Plantas de macieira originárias da França introduzidas no Brasil para fins de pesquisa foram submetidas à análise no Laboratório de Quarentena Vegetal da Embrapa Recursos Genéticos e Biotecnologia (Cenargen). Após realizar a enxertia em porta-enxertos sadios, algumas plantas apresentaram sintomas típicos de infecção por fitoplasmas. Os testes por meio da técnica de reação em cadeia da polimerase (PCR) obtiveram fragmentos de DNA do tamanho esperado para fitoplasmas. O sequenciamento de DNA revelou uma identidade de 98-99% da região 16 rDNA dos fitoplasmas encontrados com o fitoplasma 'Candidatus Phytoplasma solani'. Essa espécie de fitoplasma é responsável por perdas em pomares de maçã europeus e não foi relatada no Brasil. De acordo com a Legislação Federal de Proteção de Plantas, as plantas de macieira foram incineradas para evitar a entrada dessa praga exótica no Brasil.(AU)

RNA-mediated gene silencing signals are not graft transmissible from the rootstock to the scion in greenhouse-grown apple plants Malus sp.

RNA silencing describes the sequence specific degradation of RNA targets. Silencing is a non-cell autonomous event that is graft transmissible in different plant species. The present study is the first report on systemic acquired dsRNA-mediated gene silencing of transgenic and endogenous gene sequences in a woody plant like apple. Transgenic apple plants overexpressing a hairpin gene construct of the gusA reporter gene were produced. These plants were used as rootstocks and grafted with scions of the gusA overexpressing transgenic apple clone T355. After grafting, we observed a reduction of the gusA gene expression in T355 scions in vitro, but not in T355 scions grown in the greenhouse. Similar results were obtained after silencing of the endogenous Mdans gene in apple that is responsible for anthocyanin biosynthesis. Subsequently, we performed grafting experiments with Mdans silenced rootstocks and red leaf scions of TNR31-35 in order to evaluate graft transmitted silencing of the endogenous Mdans. The results obtained suggested a graft transmission of silencing signals in in vitro shoots. In contrast, no graft transmission of dsRNA-mediated gene silencing signals was detectable in greenhouse-grown plants and in plants grown in an insect protection tent.