Rosaceous Plants Research Articles

The species of Pteromalus Swederus in America north of Mexico with a 4:4 mandibular formula, and description of a potential biocontrol agent of the introduced pest Anthonomus rubi (Herbst) (Coleoptera: Curculionidae).

The strawberry blossom weevil, Anthonomus rubi (Herbst) (Coleoptera: Curculionidae), is native to Europe, Asia, and parts of North Africa, and has recently established in British Columbia, Canada and Washington State, USA. To determine whether any parasitoids in British Columbia parasitize this recently-established pest, A. rubi-infested buds of Rosaceous host plants were collected and reared for parasitoid emergence. Pteromalus quadridentatus Gibson n. sp. (Hymenoptera: Chalcidoidea: Pteromalidae) is described from both sexes reared as solitary ectoparasitoids of A. rubi. Males and females are partly characterized by both mandibles having four teeth (4:4 mandibular formula), which is known for only four of the other previously recorded 39 extant species of Pteromalus from the Nearctic region-P. apum (Retzius), P. cassotis Walker, P. hemileucae Gahan, and P. puparum (L.). The species of Pteromalus recorded from the Nearctic region are listed along with notes on their known mandibular dentition or other features that differentiate them from P. quadridentatus. The five species with a 4:4 mandibular formula are keyed, diagnosed and illustrated through macrophotography. Additionally, both sexes of P. quadridentatus are described more comprehensively, including variation in structure, colour and sculpture correlated with body size; the morphological species limits of P. cassotis are also discussed. Species of Pteromalus with a 4:4 mandibular formula from other regions where A. rubi is recorded are also listed and discussed, but only sufficiently to exclude them as potentially conspecific with P. quadridentatus. A phylogenetic tree constructed using Maximum Likelihood based on COI barcode sequences showed strong support for P. quadridentatus being monophyletic and sister to P. bedeguaris (Thomson). Pteromalus quadridentatus is the first parasitoid recorded from A. rubi in its invaded range, although it is still uncertain whether this parasitoid is native to North America or was introduced along with A. rubi or another host species.

The 3rd International Symposium on Fire Blight of Rosaceous Plants: a Journal of Plant Pathology special issue

The 3rd International Symposium on Fire Blight of Rosaceous Plants: a Journal of Plant Pathology special issue

Genomic insights into novel Erwinia bacteriophages: unveiling their Henunavirus membership and host infection strategies.

Erwinia amylovora, the primary causative agent of blight disease in rosaceous plants, poses a significant threat to agricultural yield worldwide, with limited effective countermeasures. The emergence of sustainable alternative agents such as bacteriophages is a promising solution for fire blight that specifically targets Erwinia. In this study, we isolated pEp_SNUABM_01 and pEa_SNUABM_55 from a South Korean apple orchard soil, analyzed their genomic DNA sequences, and performed a comprehensive comparative analysis of Hena1 in four distinct sections. This study aimed to unveil distinctive features of these phages, with a focus on host recognition, which will provide valuable insights into the evolution and characteristics of Henunavirus bacteriophages that infect plant pathogenic Erwinia spp. By elucidating the distinct genomic features of these phages, particularly in terms of host recognition, this study lays a foundation for their potential application in mitigating the risks associated with fire blight in Rosaceae plants on a global scale.

Genetic characterization of historic Norwegian Erwinia amylovora isolates by SSR-genotyping

AbstractErwinia amylovora, the causative agent of fire blight of pome fruits and other rosaceous plants belongs to the group of regulated quarantine pests. The aim of this work was to characterize the populations of E. amylovora in Norway and their geographical distribution. A total of 238 E. amylovora isolates recovered from symptomatic host plants in Norway between 1986 and 2004 were genotyped by means of a short sequence repeat (SSR) marker (ATTACAGA) on plasmid pEa29. The SSR region was amplified and amplicon size determined using fluorescent labelling and rapid, automated capillary gel electrophoresis. All isolates contained the pEa29 plasmid harbouring the investigated marker. In total, ten genotypes were identified, of which two were detected only once. The number of repeats varied from 3 to 13, with 43% of the isolates containing five repeats. Of 17 isolates collected between 1986 and 1991, all but one contained five repeats, whereas more variation was observed in isolates from the period 2000 to 2004. Most of the isolates (80%) originated from Cotoneaster bullatus, hence no relationship between genotype of the isolate and host species that it was isolated from could be detected. This historic data suggests multiple introductions of E. amylovora to Norway.

Advances in genomics and genome editing for improving strawberry (Fragaria ×ananassa).

The cultivated strawberry, Fragaria ×ananassa, is a recently domesticated fruit species of economic interest worldwide. As such, there is significant interest in continuous varietal improvement. Genomics-assisted improvement, including the use of DNA markers and genomic selection have facilitated significant improvements of numerous key traits during strawberry breeding. CRISPR/Cas-mediated genome editing allows targeted mutations and precision nucleotide substitutions in the target genome, revolutionizing functional genomics and crop improvement. Genome editing is beginning to gain traction in the more challenging polyploid crops, including allo-octoploid strawberry. The release of high-quality reference genomes and comprehensive subgenome-specific genotyping and gene expression profiling data in octoploid strawberry will lead to a surge in trait discovery and modification by using CRISPR/Cas. Genome editing has already been successfully applied for modification of several strawberry genes, including anthocyanin content, fruit firmness and tolerance to post-harvest disease. However, reports on many other important breeding characteristics associated with fruit quality and production are still lacking, indicating a need for streamlined genome editing approaches and tools in Fragaria ×ananassa. In this review, we present an overview of the latest advancements in knowledge and breeding efforts involving CRISPR/Cas genome editing for the enhancement of strawberry varieties. Furthermore, we explore potential applications of this technology for improving other Rosaceous plant species.

Biodiversity of spider mites of Ankara, with a checklist of the Tetranychinae and first report of Schizotetranychus ibericus Reck for Turkey (Acari: Prostigmata)

The samples were collected at weekly intervals between April and October from 2011 to 2014 on rosaceous plants and Quercus trees in Ankara, Turkey. Twelve spider mite species (Acari: Tetranychidae), Amphitetranychus viennensis (Zacher), Schizotetranychus ibericus Reck, Tetranychus urticae Koch, T. turkestani (Ugarov & Nikolskii), Eoteteranychus populi (Koch), E. pruni (Oudemans), E. coryli Reck, E. tiliarium (Hermann) Panonychus ulmi (Koch) (Tetranychinae), Bryobia rubrioculus (Scheuten), B. praetiosa Koch and B. sp. (Bryobiinae) were identified in Ankara. Schizotetranychus ibericus Reck, 1947 is a new record for Turkey. Tetranychus urticae and A. viennensis occurred in all the sampling localities, while S. ibericus was collected only on a Quercus sp. (Fagaceae). Tetranychus urticae Koch (Tetranychidae) was the most abundant and common species while some other species were represented by only two specimens such as Eotetranychus populi and E. tiliarum (Hermann). Apple harbored the highest number of spider mite species (9) while Prunus mahaleb and Quercus had only 2 and 1 species, respectively. The preferred hosts plants were Malus domestica (1007), Prunus domestica (749) and Prunus avium (479), while Prunus armeniaca (164), Prunus mahaleb (66) and Quercus sp. (17) were less preferred hosts. Collection data and geographical distribution of these species are provided. The checklist of the previous identified Tetranychinae species of Turkey is provided.

Highly efficient Agrobacterium rhizogenes-mediated transformation for functional analysis in woodland strawberry

BackgroundThe diploid woodland strawberry (Fragaria vesca) is an excellent model plant for investigating economically significant traits and several genetic resources within the Rosaceae family. Agrobacterium rhizogenes-mediated hairy root transformation is an alternative for exploring gene functions, especially the genes specifically expressed in roots. However, the hairy root transformation has not been established in strawberry.ResultsHere, we described an efficient and rapid hairy root transgenic system for strawberry using A. rhizogenes. Strain of A. rhizogenes MSU440 or C58C1 was the most suitable for hairy root transformation. The transformation efficiency was highest when tissues contained hypocotyls as explants. The optimal procedure involves A. rhizogenes at an optical density (OD600) of 0.7 for 10 min and co-cultivation duration for four days, achieving a transgenic efficiency of up to 71.43%. An auxin responsive promoter DR5ver2 carrying an enhanced green fluorescent protein (eGFP) marker was transformed by A. rhizogenes MSU440, thereby generating transgenic hairy roots capable of high eGFP expression in root tip and meristem of strawberry where auxin accumulated. Finally, this system was applied for functional analysis using jGCaMP7c, which could sense calcium signals. A significant upsurge in eGFP expression in the transgenic hairy roots was displayed after adding calcium chloride. The results suggested that this approach was feasible for studying specific promoters and could be a tool to analyze gene functions in the roots of strawberries.ConclusionWe established a rapid and efficient hairy root transformation in strawberry by optimizing parameters, which was adequate for promoter analysis and functional characterization of candidate genes in strawberry and other rosaceous plants.

Three new gall forming Eriophyes species (Acari: Eriophyidae) on rosaceous plants in Iran

During field surveys of eriophyoid species associated with rosaceous plants in Zagros forests of Iran, three new species were discovered and described including Eriophyes cotosarica sp. nov. from Cotoneaster hissaricus Pojark., Eriophyes cratameyeris sp. nov. from Crataegus meyeri Pojark. and Eriophyes pyrusyriaca sp. nov. from Pyrus syriaca Boiss. All species cause galls on their host plant’s leaves. In addition, a list of Eriophyes species found on Rosaceous plants in Iran and a supplementary description of Eriophyes pyri (Pagenstecher, 1857) are provided.

Target gene selection for RNAi-based biopesticides against the hawthorn spider mite, Amphitetranychus viennensis (Acari: Tetranychidae).

Recently, RNA interference (RNAi)-based biopesticide, a species-specific pest control alternative, has been deregulated and commercialized in the US and Canada. The hawthorn spider mite, Amphitetranychus viennensis Zacher, is a major pest for rosaceous plants, which has been controlled primarily by synthetic pesticides. To address the emerging resistance issues in A. viennensis, we initiated a project to develop RNAi-based biopesticides. In this study, we (i) developed a dietary RNAi system for A. viennensis using leaf disc, (ii) assessed the suitability of multiple control genes to distinguish sequence-specific silencing from non-specific effects within this RNAi system, and (iii) screened for the target gene candidates. As a result, β-Glucuronidase (GUS), an enzyme derived from E. coli and a broadly used reporter for plants is the appropriate control for A. viennensis RNAi, while green fluorescent protein (GFP), is not suitable due to its significantly higher mortality than the other controls. For target gene screening, suppression was confirmed for all the candidates, including two housekeeping genes (Vacuolar-type H + -ATPase subunit A (V-ATPase A) and Glyceraldehyde 3-phosphate dehydrogenase, (GAPDH)), and three genes associated with development (ATP-dependent RNA Helicase DDX3Y (Belle), CREB-binding protein (CBP), and Farnesoic acid O-methyltransferase (FaMet)). Knocking down of V-ATPase A resulted in the highest mortality (~ 90%) and reduced fecundity (over 90%) than other candidates. As for the genes associated with development, suppression of Belle and CBP, led to approximately 65% mortality, as well as 86% and 40% reduction in fecundity, respectively. Silencing of FaMet, however, had negligible biological impacts on A. viennensis. The combined efforts not only establish an effective dsRNA delivery method, but also provide potential target genes for RNAi-based biopesticides against A. viennensis, a devastating invasive pest for fruit trees and woody ornamental plants throughout Asia and Europe. © 2023 Society of Chemical Industry.

Isolation and characterization of Priestia megaterium KD7 for the biological control of pear fire blight.

Erwinia amylovora is a plant pathogen that causes fire blight disease in Rosaceous plants, such as pear and apple. To develop an effective biocontrol method to suppress E. amylovora, a total of 16 bacteria were isolated from pear orchard soil in China and screened for antagonistic activity in vitro. Among them, 9 isolates that exhibited antagonistic activity against E. amylovora were identified, including Bacillus atrophaeus, Priestia megaterium (previously known as Bacillus megaterium) and Serratia marcescens based on the partial 16S rDNA sequence analysis and similarity search. The plate confrontation experiments showed that strain 8 (P. megaterium strain KD7) had strong antagonistic activity against E. amylovora. The methanolic extract from cell-free supernatant of strain KD7 displayed high antibacterial activities against E. amylovora. Furthermore, the active compounds of strain KD7 were separated by thin layer chromatography (TLC) and the amino acids were detected by the presence of a spot with retention factor (Rf) of 0.71. Next, three lipopeptides were identified with high-resolution mass spectrometry (HRMS), including C13-surfactin [M+H]+ at m/z 1008.14, C15-surfactin [M+H]+ at m/z 1036.50, and C14-iturin A [M+H]+ at m/z 1043.17. Strain KD7 showed multiple antibiotic resistance, such as ampicillin, erythromycin, penicillin and tetracycline. The detached pear leaves, twigs and fruits assay showed that both protective and curative action with strain KD7 had the ability to decrease the development of fire blight. Taken together, P. megaterium strain KD7 is a potential effective biocontrol agent against fire blight.

Development and Fecundity of Oriental Fruit Moth (Lepidoptera: Tortricidae) Reared on Various Concentrations of Amygdalin

Grapholita molesta (Busck) (Lepidoptera: Tortricidae), Oriental fruit moth (OFM), attacks fruits and shoots of the economically important trees in Rosaceae. Amygdalin is a cyanogenic glucoside of rosaceous plants that may be related to the seasonal patterns of infestation in many pests. The amygdalin concentration of fruits and shoots of peach, pear, and apple varies over the growing season. However, the relationship between the amygdalin concentration and G. molesta performance has not been reported. Here, we measured the performance (feeding, growth, development, and fecundity) of G. molesta larvae (as subsequent adults) reared on artificial diets with six amygdalin concentrations (0, 3, 6, 12, 24, and 48 mg/g), and we then calculated the population parameters. We found that these different concentrations of amygdalin affected the developmental time and fecundity, except for the proportion of larvae feeding on the diet and the survival rates of larvae and pupae. When compared with the control diet without amygdalin, diets with 3 or 6 mg/g (low and moderate concentrations) of amygdalin shortened developmental times and increased the number of eggs laid by females; however, a diet with 12 mg/g (moderate concentration) of amygdalin only increased the number of eggs laid by females and did not affect the larval and pupal developmental rate. A diet with 48 mg/g (high concentration) of amygdalin prolonged developmental times and reduced the number of eggs laid by females when compared with the control diet without amygdalin. Furthermore, the intrinsic rate of increase (rm) for insects reared on diets with 3 or 6 mg/g (low and moderate concentrations) of amygdalin versus the control diet without amygdalin showed a slightly improved population growth. However, this increase in the rm value did not persist over ten successive generations of rearing on the same diet. We concluded that the diet with 6 mg of amygdalin per g of diet can enhance the performance and population growth of G. molesta, but the effects of amygdalin are concentration-dependent.

Biological Control Potential of Penicillium brasilianum against Fire Blight Disease.

Erwinia amylovora is a causative pathogen of fire blight disease, affecting apple, pear, and other rosaceous plants. Currently, management of fire blight relies on cultural and chemical practices, whereas it has been known that few biological resources exhibit disease control efficacy against the fire blight. In the current study, we found that an SFC20201208-M01 fungal isolate exhibits antibacterial activity against E. amylovora TS3128, and the isolate was identified as a Penicillium brasilianum based on the β-tubulin (BenA) gene sequence. To identify active compounds from the P. brasilianum culture, the culture filtrate was partitioned with ethyl acetate and n-butanol sequentially. From the ethyl acetate layer, we identified two new compounds (compounds 3-4) and two known compounds (compounds 1-2) based on spectroscopic analyses and comparison with literature data. Of these active compounds, penicillic acid (1) exhibited promising antibacterial activity against E. amylovora TS3128 with a minimal inhibitory concentration value of 25 μg/ml. When culture filtrate and penicillic acid (125 μg/ml) were applied onto Chinese pearleaf crab apple seedlings prior to inoculation of E. amylovora TS3128, the development of fire blight disease was effectively suppressed in the treated plants. Our results provide new insight into the biocontrol potential of P. brasilianum SFC20201208-M01 with an active ingredient to control fire blight.

Antagonistic effect of Leuconostoc mesenteroides on grapevine crown gall and fire blight

ABSTRACT Grapevine crown gall, caused by Allorhizobium vitis (A. vitis) and fire blight, caused by Erwinia amylovora (E. amylovora) of rosaceous plants, have been recognized as serious bacterial diseases and responsible for major crop losses in grapevine (Vitis vinifera L.), pear (Pyrus communis L.) and apple (Malus domestica Borkh.) worldwide. To develop an effective biocontrol method against the two bacterial pathogens, lactic acid bacteria (LAB) isolated from agricultural irrigation canals were tested as biocontrol agents. Thirty-two bacterial isolates were screened for their in vitro antagonistic activity. Four isolates (MS-1, MS-2, MS-3, and MS-4), identified by partial sequencing of 16S rRNA as Leuconostoc mesenteroides subsp. Jonggajibkimchii, were found to significantly inhibit the growth of A. vitis and E. amylovora in vitro. The biocontrol efficiency of these strains was tested against E. amylovora on immature pear fruits under laboratory conditions, and against A. vitis in tomato (Solanum lycopersicum L.) and kalanchoe (Kalanchoe daigremontiana) plants, under greenhouse conditions. Results showed that all selected antagonists were able to suppress fire blight disease on immature pear fruits. On tomato plants, the MS-1 and MS-2 strains were able to reduce the A. vitis infection by 82% and 99%, respectively. Similarly, the MS-2 strain reduced gall formation on kalanchoe plants by 99%. We concluded that Leuconostoc mesenteroides subsp. Jonggajibkimchii strains could serve as a potential biocontrol agent against fire blight and grapevine crown gall diseases.

Chemical composition and antibacterial activity of Moroccan Artemisia mesatlantica and A. absinthium essential oils against fire blight caused by Erwinia amylovora

The aim of this study was to find an alternative to chemical products used in the control of fire blight disease of rosaceous plants. In this work, essential oils of two Moroccan Artemisia species: Artemisia mesatlantica and Artemisia absinthium were screened for their antibacterial activity against Erwinia amylovora the causal agent of this disease. The EOs extraction was carried out by water distillation method in Clevenger- type apparatus and analyzed by gas Chromatography-Flame Ionization Detector (GC/FID) and by Gas Chromatography-Mass Spectrometry (GC/MS). The antibacterial activity of extracted EOs was tested in vitro based on the agar diffusion method and broth microdilution method and in vivo on detached immature pear fruits. The results revealed that the yields of extracted EOs were 1.25 and 0.58% respectively for A. mesatlantica and A. absinthium. The chemical composition analysis showed that investigated EOs were characterized with a high amount of β-Thujone (40.42% for A. mesatlantica and 36.25% for A. absinthium). Both EOs have a similar MIC value (2.08%). A. absinthium EOs were found to be more effective than A. mesatlantica EOs since they show the lowest MBC. These results indicate that EOs of A. absinthium and A. mesatlantica contained compounds with antibacterial potential against Erwinia amylovora. In the bioassay test, the application of A. absinthium EOs on detached immature pear fruits infected by E. amylovora resulted in total suppression of disease severity. Such natural products may represent a sustainable alternative to the use of chemical pesticides.

The RNA-Binding Protein ProQ Impacts Exopolysaccharide Biosynthesis and Second Messenger Cyclic di-GMP Signaling in the Fire Blight Pathogen Erwinia amylovora.

Erwinia amylovora is a plant-pathogenic bacterium that causes fire blight disease in many economically important plants, including apples and pears. This bacterium produces three exopolysaccharides (EPSs), amylovoran, levan, and cellulose, and forms biofilms in host plant vascular tissues, which are crucial for pathogenesis. Here, we demonstrate that ProQ, a conserved bacterial RNA chaperone, was required for the virulence of E. amylovora in apple shoots and for biofilm formation in planta. In vitro experiments revealed that the deletion of proQ increased the production of amylovoran and cellulose. Prc is a putative periplasmic protease, and the prc gene is located adjacent to proQ. We found that Prc and the associated lipoprotein NlpI negatively affected amylovoran production, whereas Spr, a peptidoglycan hydrolase degraded by Prc, positively regulated amylovoran. Since the prc promoter is likely located within proQ, our data showed that proQ deletion significantly reduced the prc mRNA levels. We used a genome-wide transposon mutagenesis experiment to uncover the involvement of the bacterial second messenger c-di-GMP in ProQ-mediated cellulose production. The deletion of proQ resulted in elevated intracellular c-di-GMP levels and cellulose production, which were restored to wild-type levels by deleting genes encoding c-di-GMP biosynthesis enzymes. Moreover, ProQ positively affected the mRNA levels of genes encoding c-di-GMP-degrading phosphodiesterase enzymes via a mechanism independent of mRNA decay. In summary, our study revealed a detailed function of E. amylovora ProQ in coordinating cellulose biosynthesis and, for the first time, linked ProQ with c-di-GMP metabolism and also uncovered a role of Prc in the regulation of amylovoran production. IMPORTANCE Fire blight, caused by the bacterium Erwinia amylovora, is an important disease affecting many rosaceous plants, including apple and pear, that can lead to devastating economic losses worldwide. Similar to many xylem-invading pathogens, E. amylovora forms biofilms that rely on the production of exopolysaccharides (EPSs). In this paper, we identified the RNA-binding protein ProQ as an important virulence regulator. ProQ played a central role in controlling the production of EPSs and participated in the regulation of several conserved bacterial signal transduction pathways, including the second messenger c-di-GMP and the periplasmic protease Prc-mediated systems. Since ProQ has recently been recognized as a global posttranscriptional regulator in many bacteria, these findings provide new insights into multitiered regulatory mechanisms for the precise control of virulence factor production in bacterial pathogens.

Study of Bacillus velezensis OEE1 potentialities in the biocontrol against Erwinia amylovora, causal agent of fire blight disease of rosaceous plants

Erwinia amylovora is the causal agent of fire blight disease affecting rosaceous plants. The present study focuses on the potential of Bacillus velezensis as a promising biological control agent toward this phytopathogenic bacterium. B. velezensis OEE1 showed the most effective antibacterial activity (80 %) among 165 tested bacterial strains under in vitro conditions against E. amylovora. Following the extraction of the active molecules, the antibacterial activity was detected in butanol extract. A separation of the active compounds using thin layer chromatography (TLC) of the butanol extract of the culture filtrate of B. velezensis OEE1 indicate the presence of amino acids by the presence of a spot with a frontal ratio (Rf) of 0.73. B. velezensis OEE1 also showed high levels of multi-resistance to penicillin, ampicillin, kanamycin, ciprofloxacin, amoxicillin and streptomycin. Biological and chemical treatments (by B. subtilis Y1336 and copper oxide Cu2O respectively) were used to compare the efficacy of the treatment with the antagonistic bacterium OEE1. The results indicated that both protective and curative treatments with antagonistic bacterium were effective to reduce the disease progression. The curative method was generally significantly more efficient than the protective method for tests on detached tissues. The efficacy of B. velezensis OEE1 to reduce E. amylovora was evaluated also in greenhouse and under field conditions. B. velezensis OEE1 was able to improve the collar diameter and the number of leaves of healthy pear plants of the variety “Alexandrine” in greenhouse conditions. Experiments in greenhouse and under field conditions revealed that B. velezensis OEE1 significantly decreased the final mean disease severity index (FMS) and the percentage of dead plants (PDP). This study has demonstrated that B. velezensis is considered as a future potential new biocontrol agent for controlling fire blight disease.

Expression of the Type III Secretion System Genes in Epiphytic Erwinia amylovora Cells on Apple Stigmas Benefits Endophytic Infection at the Hypanthium.

Erwinia amylovora causes fire blight on rosaceous plants. One of the major entry points of E. amylovora into hosts is flowers, where E. amylovora proliferates epiphytically on stigmatic and hypanthium surfaces and, subsequently, causes endophytic infection at the hypanthium. The type III secretion system (T3SS) is an important virulence factor in E. amylovora. Although the role of T3SS during endophytic infection is well characterized, its expression during epiphytic colonization and role in the subsequent infection is less understood. Here, we investigated T3SS gene expression in epiphytic E. amylovora on stigma and hypanthium of apple flowers under different relative humidities (RH). On stigma surfaces, T3SS was expressed in a high percentage of E. amylovora cells, and its expression promoted epiphytic growth. On hypanthium surfaces, however, T3SS was expressed in fewer E. amylovora cells than on the stigma, and displayed no correlation with epiphytic growth, even though T3SS expression is essential for infection. E. amylovora cells grown on stigmatic surfaces and then flushed down to the hypanthium displayed a higher level of T3SS expression than cells grown on the hypanthium surface alone. Furthermore, E. amylovora cells precultured on stigma had a higher potential to infect flowers than E. amylovora cells precultured in a T3SS-repressive medium. This suggests that T3SS induction during the stigmatic epiphytic colonization may be beneficial for subsequent infection. Finally, epiphytic expression of T3SS was influenced by RH. Higher percentage of stigmatic E. amylovora cells expressed T3SS under high RH than under low RH.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

High-quality genome assembly of 'Cuiguan' pear (Pyrus pyrifolia) as a reference genome for identifying regulatory genes and epigenetic modifications responsible for bud dormancy

Dormancy-associated MADS-box (DAM) genes serve as crucial regulators of the endodormancy cycle in rosaceous plants. Although pear DAM genes have been identified previously, the lack of a high-quality reference genome and techniques to study gene function have prevented accurate genome-wide analysis and functional verification of such genes. Additionally, the contribution of other genes to the regulation of endodormancy release remains poorly understood. In this study, a high-quality genome assembly for 'Cuiguan' pear (Pyrus pyrifolia), which is a leading cultivar with a low chilling requirement cultivated in China, was constructed using PacBio and Hi-C technologies. Using this genome sequence, we revealed that pear DAM genes were tandemly clustered on Chr8 and Chr15 and were differentially expressed in the buds between 'Cuiguan' and the high-chilling-requirement cultivar 'Suli' during the dormancy cycle. Using a virus-induced gene silencing system, we determined the repressive effects of DAM genes on bud break. Several novel genes potentially involved in the regulation of endodormancy release were identified by RNA sequencing and H3K4me3 chromatin immunoprecipitation sequencing analyses of 'Suli' buds during artificial chilling using the new reference genome. Our findings enrich the knowledge of the regulatory mechanism underlying endodormancy release and chilling requirements and provide a foundation for the practical regulation of dormancy release in fruit trees as an adaptation to climate change.

First Report of Anthracnose of Papaya (Carica papaya L.) Caused by Colletotrichum siamense in China.

Papaya (Carica papaya L.) is a rosaceous plant widely grown in China, which is economically important. Anthracnose caused by Colletotrichum sp. is an important postharvest disease, which severely affects the quality of papaya fruits (Liu et al., 2019). During April 2020, some mature papaya fruits with typical anthracnose symptoms were observed in Fusui, Nanning, Guangxi, China with an average of 30% disease incidence (DI) and over 60% DI in some orchards. Initial symptoms of these papayas appeared as watery lesions, which turned dark brown, sunken, with a conidial mass appearing on the lesions under humid and warm conditions. The disease severity varied among fruits, with some showing tiny light brown spots, and some ripe fruits presenting brownish, rounded, necrotic and depressed lesions over part of their surface. Samples from two papaya plantations(107.54°E, 22.38°N) were collected, and brought to the laboratory. Symptomatic diseased tissues were cut into 5 × 5 mm pieces, surface sterilized with 2% (v/v) sodium hypochlorite for 1 minute, and rinsed three times with sterilized water. The pieces were then placed on potato dextrose agar (PDA). After incubation at 25°C in the dark for one week, colonies with uniform morphology were obtained. The aerial mycelium on PDA was white on top side, and concentric rings of salmon acervuli on the underside. A gelatinous layer of spores was observed on part of PDA plates after 7 days at 28°C. The conidia were elliptical, aseptate and hyaline (Zhang et al., 2020). The length and width of 60 conidia were measured for each of the two representative isolates, MG2-1 and MG3-1, and these averaged 13.10 × 5.11 μm and 14.45 × 5.95 μm. DNA was extracted from mycelia of these two isolates with the DNA secure Plant Kit (TIANGEN, Biotech, China). The internal transcribed spacer (ITS), partial actin (ACT), calmodulin (CAL), chitin synthase (CHS), β-tubulin 2 (TUB2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) regions were amplified by PCR and sequenced. The sequences were deposited into GenBank with accessions MT904003, MT904004, and MT898650 to MT898659. BLASTN analyses against the GenBank database showed that they all had over 99% identity to the type strain of Colletotrichum siamense isolate ICMP 18642 (GenBank accession numbers JX010278, GQ856775, JX009709, GQ856730, JX010410, JX010019) (Weir et al., 2012). A phylogenetic tree based on the combined ITS, ACT, CAL, CHS, TUB2 and GAPDH sequences using the Neighbor-joining algorithm also showed that the isolates were C. siamense. Pathogenicity tests were conducted on 24 mature, healthy and surface-sterilized papaya fruits. On 12 papaya fruits, three well separated wounded sites were made for inoculation, and for each wounded site, six adjacent pinhole wounds were made in a 5-mm-diameter circular area using a sterilized needle. A 10 µl aliquot of 1 × 106 conidia/ml suspension of each of the isolates (MG2-1 and MG3-1) was inoculated into each wound. For each isolate, there were six replicate fruits. The control fruits were inoculated with sterile distilled water. The same inoculation was applied to 12 non-wound papaya fruits. Fruits were then placed in boxes which were first washed with 75% alcohol and lined with autoclaved filter paper moistened with sterilized distilled water to maintain high humidity. The boxes were then sealed and incubated at 28°C. After 10 days, all the inoculated fruits showed symptoms, while the fruits that were mock inoculated were without symptoms. Koch's postulates were fulfilled by re-isolation of C. siamense from diseased fruits. To our knowledge, this is the first report of C. siamense causing anthracnose of papaya in China. This finding will enable better control of anthracnose disease caused by C. siamense on papaya.

Comparison of Bacterial Community of Healthy and Erwinia amylovora Infected Apples.

Fire blight disease, caused by Erwinia amylovora, could damage rosaceous plants such as apples, pears, and raspberries. In this study, we designed to understand how E. amylovora affected other bacterial communities on apple rhizosphere; twig and fruit endosphere; and leaf, and fruit episphere. Limited studies on the understanding of the microbial community of apples and changes the community structure by occurrence of the fire blight disease were conducted. As result of these experiments, the infected trees had low species richness and operational taxonomic unit diversity when compared to healthy trees. Rhizospheric bacterial communities were stable regardless of infection. But the communities in endosphere and episphere were significanlty affected by E. amylovora infection. We also found that several metabolic pathways differ significantly between infected and healthy trees. In particular, we observed differences in sugar metabolites. The finding provides that sucrose metabolites are important for colonization of E. amylovora in host tissue. Our results provide fundamental information on the microbial community structures between E. amylovora infected and uninfected trees, which will contribute to developing novel control strategies for the fire blight disease.