First Report of Lasiodiplodia pseudotheobromae Causing Necrosis and Canker Disease on Pear Tree in China.

Pear is popular among people, which is an important pillar industry in China. In March of 2023, dark brown necrotic lesions were discovered on the trunks of Pyrus pyrofolia cv. Osmanthus pear in orchard, Liuzhou City, Guangxi Zhuang Autonomous Region. In August, field investigation and sample collection were conducted in orchard. Forty pear trees were selected for symptomatic observation, which of 21 had lesions ranging from 10 to 24 per tree, and 19 with 1 to 8 lesions, respectively. To isolate the pathogen, small tissue pieces of 3 diseased pear trunk samples were disinfected with 75% ethanol for 1 minute, rinsed with sterile water, and dried with filter paper. The tissue pieces were placed on potato dextrose agar (PDA) plates and cultured in a dark incubator at 25℃. Six isolates with the similar morphology were obtained. One of the six isolates was randomly selected as the representative strain and named as GX-3. Mycelium grows with an average rate of 4.26 cm/d. The hypha is highly aerial, and is initially white and then turns black. Subsequently, pycnidia formed and secreted black mucus on the PDA medium after 28 days. The immature conidia were ellipsoid, colorless, hyaline, and unicellular, mostly becoming brown bicellular with longitudinal stripes at maturity. The conidial size was 22.5 to 32.6×12.1 to 19.7µm, and the average size was 28.4±2.3×16.7±2.0 µm (n=50), respectively. GX-3 colony morphology was consistent with that of Lasiodiplodia pseudotheobromae (Alves et al.2008). For molecular identification, the internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (TEF1-α), and ß-tubulin regions were amplified using the primers ITS1/4, EF1-728F/986R, and Bt2a/Bt2b, respectively (White et al.1990; Carbone and Kohn 1999; Glass and Donaldson 1995). The obtained sequences of GX-3 were deposited in NCBI with Accession numbers OR655421, OR661231, and OR661230, respectively. The sequences of ITS, TEF1-α, and ß-tubulin from GX-3 are 99.44%、99.67% and 99.78% identities with those of L. pseudotheobromae CBS 447.62, respectively. The phylogenetic analysis was performed by maximum likelihood method, revealing that GX-3 is closely clustered with the isolates of L. pseudotheobromae. Therefore, the GX-3 strain was identified as L. pseudotheobromae. GX-3 was further analyzed for its pathogenicity on pear. Firstly, the GX-3 mycelium plugs and spraying spore suspension with the concentration of 1×107 conidia/ml were applied on the stems of 4-month-old healthy birch-leaf pear (Pyrus betulifolia Bunge) potted seedlings by acupuncture needle method, meanwhile PDA and sterile water were used as controls. After 3 days of inoculation, stem surface of the birch-leaf pear exhibited dark brown lesions with slight surface depression, obvious dryness, and canker symptoms, while the control treatment showed no symptoms. The GX-3 was also inoculated on in vitro branches of 'Hosui', 'Hongxiangsu', 'Bodoqing' and 'Xuehua', showing dark brown canker lesions. The same pathogen can be successfully isolated from diseased stems and branches but not from the controls, which accomplishes Koch's postulates. L. pseudotheobromae has been widely reported that it can cause rot and canker on apple, walnut, hackberry, and so on (Xue et al. 2019; Wang et al. 2023; Liang et al. 2020). This is the first report of necrosis and canker disease caused by L. pseudotheobromae on pear in China, which is a potential threat to pear industry.

Genome­wide characterization of the Gα subunit gene family in Rosaceae and expression analysis of PbrGPAs under heat stress.

The Gα subunit is an important component of the heterotrimeric G-protein complex and an integral component of several signal transduction pathways. It plays crucial roles in the diverse processes of plant growth and development, including the response to abiotic stress, regulation of root development, involvement in stomatal movement, and participation in hormone responses, which have been well investigated in many species. However, no comprehensive analysis has identified and explored the evolution, expression pattern characteristics and heat stress response of the Gα subunit genes in Rosaceae. In this study, 52 Gα subunit genes were identified in eight Rosaceae species; these genes were divided into three subfamilies (I, II, and III) based on their phylogenetic, conserved motif, and structural characteristics. Whole genome and dispersed duplication events were found to have contributed significantly to the expansion of the Gα subunit gene family, and purifying selection to have played a key role in the evolution of Gα subunit genes. An expression analysis identified some PbrGPA genes that were highly expressed in leaf, root, and fruit, and exhibited diverse spatiotemporal expression models in pear. Under abiotic stress conditions, the mRNA transcript levels of PbrGPA genes were up-regulated in response to high temperature treatment in leaves. Furthermore, three Gα subunit genes were shown to be located in the plasma membrane and nucleus in pear. In conclusion, the study of the Gα subunit gene family will help us to better understand its evolutionary history and expression patterns, while facilitating further investigations into the function of the Gα subunit gene in response to heat stress.

Genome-Wide Identification and Comparative Analysis of the ASR Gene Family in the Rosaceae and Expression Analysis of PbrASRs During Fruit Development.

The members of the Abscisic Acid (ABA) Stress and Ripening gene family (ASR) encode a class of plant-specific proteins with ABA/WDS domains that play important roles in fruit ripening, abiotic stress tolerance and biotic stress resistance in plants. The ASR gene family has been widely investigated in the monocotyledons and dicotyledons. Although the genome sequence is already available for eight fruit species of the Rosaceae, there is far less information about the evolutionary characteristics and the function of the ASR genes in the Rosaceae than in other plant families. Twenty-seven ASR genes were identified from species in the Rosaceae and divided into four subfamilies (I, II, III, and IV) on the basis of structural characteristics and phylogenetic analysis. Purifying selection was the primary force for ASR family gene evolution in eight Rosaceae species. qPCR experiments showed that the expression pattern of PbrASR genes from Pyrus bretschneideri was organ-specific, being mainly expressed in flower, fruit, leaf, and root. During fruit development, the mRNA abundance levels of different PbrASR genes were either down- or up-regulated, and were also induced by exogenous ABA. Furthermore, subcellular localization results showed that PbrASR proteins were mainly located in the nucleus and cytoplasm. These results provide a theoretical foundation for investigation of the evolution, expression, and functions of the ASR gene family in commercial fruit species of the Rosaceae family.

Transcriptome analysis reveals the regulation of metabolic processes during the post-harvest cold storage of pear.

Pear is a traditional and economically fruit tree worldwide. With the development of the pear industry, pear fruit post-harvest preservation techniques have become very important. Among them, low temperature preservation technology is most widely used, but the molecular mechanism underlying this process is still unclear. To better understand this, RNA-seq was performed on samples collected at different time points with increasing storage time. Here, 19,610 differentially expressed genes were obtained and annotated into 51 GO terms and 26 KEGG-defined significantly overrepresented pathways. 2475 transcription factors belonging to 50 different families were identified with increasing storage time. Ethylene content increased with storage time and was the highest at 105 days of fruit storage. Accordingly, integrative analysis of gene expression revealed that 14 unigenes were related to the ethylene metabolic pathway. This study provides valuable resources to investigate the genetics of the ethylene metabolic pathways and improve pear storage and preservation technology.

Identification of hexokinase family members in pear (Pyrus × bretschneideri) and functional exploration of PbHXK1 in modulating sugar content and plant growth.

Hexokinase (HXK) is a multifunctional protein that serves as a sugar sensor for glucose signaling and a catalyst for glycolysis. It has been well studied in many species, however, there is far less information about this family in pear. To investigate the roles of HXK in the growth and development of pear fruit, we performed a genome-wide analysis and identified the HXK gene family members in pear. In addition, we functionally characterized a glucose sensor gene, PbHXK1, in P. bretschneideri. In total, 10 HXK genes were identified in pear, and a multiple sequence alignment and phylogenetic analysis showed that PbHXK1 is a Type B HXK that contains four conserved domains, phosphate 1 and 2, sugar binding and adenosine, which are specific to plant HXKs and essential for enzymatic functions. A qRT-PCR analysis revealed that the relative expression levels of PbHXK1 were negatively correlated with sugar content but significantly positively correlated with HXK activity during pear fruit development. Furthermore, the overexpression of PbHXK1 in tomatoes significantly enhanced the HXK activity and decreased the sugar content. In addition, the growth of transgenic tomato plants overexpressing PbHXK1 was inhibited, leading to shortened internodes and smaller leaves. Thus, in pear, PbHXK1 encodes HXK, which regulated the sugar content in fruit and affected the growth and development of plants.