Uncovering ghost introgression through genomic analysis of a distinct eastern Asian hickory species.

Ghost introgression, or the transfer of genetic material from extinct or unsampled lineages to sampled species, has attracted much attention. However, conclusive evidence for ghost introgression, especially in plant species, remains scarce. Here, we newly assembled chromosome-level genomes for both Carya sinensis and Carya cathayensis, and additionally re-sequenced the whole genomes of 43 C. sinensis individuals as well as 11 individuals representing 11 diploid hickory species. These genomic datasets were used to investigate the reticulation and bifurcation patterns within the genus Carya (Juglandaceae), with a particular focus on the beaked hickory C. sinensis. By combining the D-statistic and BPP methods, we obtained compelling evidence that supports the occurrence of ghost introgression in C. sinensis from an extinct ancestral hickory lineage. This conclusion was reinforced through the phylogenetic network analysis and a genome scan method VolcanoFinder, the latter of which can detect signatures of adaptive introgression from unknown donors. Our results not only dispel certain misconceptions about the phylogenetic history of C. sinensis but also further refine our understanding of Carya's biogeography via divergence estimates. Moreover, the successful integration of the D-statistic and BPP methods demonstrates their efficacy in facilitating a more precise identification of introgression types.

Transcriptome profile of pecan scab resistant and susceptible trees from a pecan provenance collection.

Pecan scab is a devastating disease that causes damage to pecan (Carya illinoinensis (Wangenh.) K. Koch) fruit and leaves. The disease is caused by the fungus Venturia effusa (G. Winter) and the main management practice for controlling the disease is by application of fungicides at 2-to-3-week intervals throughout the growing season. Besides disease-related yield loss, application of fungicides can result in considerable cost and increases the likelihood of fungicide resistance developing in the pathogen. Resistant cultivars are available for pecan growers; although, in several cases resistance has been overcome as the pathogen adapts to infect resistant hosts. Despite the importance of host resistance in scab management, there is little information regarding the molecular basis of genetic resistance to pecan scab.The purpose of this study was to elucidate mechanisms of natural pecan scab resistance by analyzing transcripts that are differentially expressed in pecan leaf samples from scab resistant and susceptible trees. The leaf samples were collected from trees in a provenance collection orchard that represents the natural range of pecan in the US and Mexico. Trees in the orchard have been exposed to natural scab infections since planting in 1989, and scab ratings were collected over three seasons. Based on this data, ten susceptible trees and ten resistant trees were selected for analysis. RNA-seq data was collected and analyzed for diseased and non-diseased parts of susceptible trees as well as for resistant trees. A total of 313 genes were found to be differentially expressed when comparing resistant and susceptible trees without disease. For susceptible samples showing scab symptoms, 1,454 genes were identified as differentially expressed compared to non-diseased susceptible samples. Many genes involved in pathogen recognition, defense responses, and signal transduction were up-regulated in diseased samples of susceptible trees, whereas differentially expressed genes in pecan scab resistant samples were generally down-regulated compared to non-diseased susceptible samples.Our results provide the first account of candidate genes involved in resistance/susceptibility to pecan scab under natural conditions in a pecan orchard. This information can be used to aid pecan breeding programs and development of biotechnology-based approaches for generating pecan cultivars with more durable scab resistance.

Infection mechanism of Botryosphaeria dothidea and the disease resistance strategies of Chinese hickory (Carya cathayensis).

Botryosphaeria dothidea is the main fungal pathogen responsible for causing Chinese hickory (Carya cathayensis) dry rot disease, posing a serious threat to the Chinese hickory industry. Understanding the molecular basis of B. dothidea infection and the host's resistance mechanisms is crucial for controlling and managing the ecological impact of Chinese hickory dry rot disease. This study utilized ultrastructural observations to reveal the process of B. dothidea infection and colonization in Chinese hickory, and investigated the impact of B. dothidea infection on Chinese hickory biochemical indicators and plant hormone levels. Through high-throughput transcriptome sequencing, the gene expression profiles associated with different stages of B. dothidea infection in Chinese hickory and their corresponding defense responses were described. Additionally, a series of key genes closely related to non-structural carbohydrate metabolism, hormone metabolism, and plant-pathogen interactions during B. dothidea infection in Chinese hickory were identified, including genes encoding DUF, Myb_DNA-binding, and ABC transporter proteins. These findings provide important insights into elucidating the pathogenic mechanisms of B. dothidea and the resistance genes in Chinese hickory.

What's in my Pot? Six Colletotrichum Species Causing Anthracnose in Brazilian Pecan Orchards.

Pecan (Carya illinoinensis) is one important exotic forest crop cultivated in South America, specifically in Brazil, Uruguay, and Argentina. However, diseases such as anthracnose, favored by high humidity conditions and high summer temperatures, make its cultivation difficult, causing important loss to pecan farmers. This study used morphological and molecular approaches to identify the Colletotrichum species causing anthracnose in pecan plantations in Southern Brazil. The isolates obtained from pecan fruits with anthracnose symptoms were grouped through quantitative morphological characteristics into three distinct morphotypes. Molecular analysis of nuclear genes allowed the identification of six species of Colletotrichum causing anthracnose in pecan: C. nymphaeae, C. fioriniae, C. gloeosporioides, C. siamense, C. kahawae, and C. karsti. Three of these species are reported for the first time as causal agents of anthracnose in pecan. Therefore, these results provide an important basis for the adoption and/or development of anthracnose management strategies in pecan orchards cultivated in southern Brazil and neighboring countries.

Scab intensity in pecan trees in relation to hedge-pruning methods.

Pecan is a valuable nut crop cultivated in the southeastern US. Among the major yield-limiting factors in the region is scab, caused by the plant pathogenic fungus Venturia effusa. Managing scab in tall trees (15 to 25+ m) in pecan orchards is challenging due to the limitations of getting sufficient spray coverage throughout the canopy. We explored the effects of hedge-pruning on scab in three orchards: 14 m tall cv. Desirable trees winter hedge-pruned on alternate sides to 11 m (site 1), 18 m tall cv. Stuart trees hedge-pruned on both sides simultaneously to 11 m (site 2), and 15 m tall cv. Caddo trees winter hedge-pruned in winter vs. summer to 11 m (site 3). At site 1 and 2 hedge-pruned trees were compared to non-pruned control trees. All trees received recommended fungicide applications to control scab via air-blast sprayer. Disease incidence and/or severity was assessed at different sample heights on shoots, foliage and fruit during three seasons (2020, 2021, and 2022). At site 1 the hedge pruned trees often had significantly or numerically more severe scab on foliage and fruit compared to the control trees, although the differences were mostly small. The frequency of mature fruit with scab severity <10% was greatest on control trees in 2021 and 2022. At site 2, there were few differences between hedge-pruned and control trees (on fruit, scab severity was either significantly less on hedge-pruned trees, or not different to the control), but the frequency of mature fruit with scab severity <10% was consistently greatest on hedge-pruned trees. At site 3, scab intensity was low, and there were no significant differences in scab severity between winter- and summer-pruning treatments. At sites 1 and 2 there was generally more severe scab at greater sample heights compared to low in the canopy. At site 3 there was little effect of height on disease. The benefit of hedge-pruning likely increases with tree height in scab-susceptible cultivars. If a tree is >~15 m tall, a greater proportion of the fruit will be within reach of efficacious spray coverage from air-blast sprayers.

Quantitative determination of specialised metabolites in different parts of Juglans regia Linn. and Carya illinoinensis (Wangenh.) K. Koch by using UHPLC-DAD-QTOF-MS/MS.

INTRODUCTION: Juglans regia Linn. and Carya illinoinensis (Wangenh.) K. Koch are nut-producing plant species of the Juglandaceae family. Bioactive compounds like naphthoquinones, tetralones, and diarylheptanoids are dominant in these species. OBJECTIVE: The study aimed to develop and validate a fast and sensitive analytical method by ultrahigh-performance liquid chromatography diode array detection mass spectrometry (UHPLC-DAD-MS) for quantification and identification of bioactive compounds in fruit pericarps and leaves of J. regia and C. illinoinensis collected from two different states of north India. METHODOLOGY: The dried pericarps of J. regia and C. illinoinensis (500 mg) were extracted with ethyl acetate-methanol (50:50 v/v, 20 mL, 50°C, 30 min) by ultrasonication and analysed by ultrahigh-performance liquid chromatography diode array detection quadrupole time-of-flight tandem mass spectrometry (UHPLC-DAD-QTOF-MS/MS) for qualitative and quantitative examination of phytoconstituents. The method was validated according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human use (ICH) guidelines for linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ). RESULTS: Here, we report the quantification of dihydrophaseic acid (1), 4,5-dihydroxytetralone (2), 4,8-dihydroxytetralone (3), 5,8-dihydroxy-3-methoxytetralone (4), and juglanin A (5) in the pericarps and leaves of J. regia and C. illinoinensis. Furthermore, using the hyphenated analytical method, a total of 62 compounds were tentatively characterised in different samples. The principal component analysis (PCA) showed diversity between the analysed sample's composition. Also, the study evaluated the variation of bioactive compounds among different parts of J. regia and C. illinoinensis collected from different regions of northern India by UHPLC-DAD-QTOF-MS/MS. CONCLUSION: The developed method is simple, rapid, and selective for the identification and characterisation of bioactive molecules.

Variations in amino acids caused by drought stress mediate the predisposition of Carya cathayensis to Botryosphaeria canker disease.

Abiotic stresses can affect the outcome of plant-pathogen interactions, mostly by predisposing the host plant to infection; however, the crosstalk between pathogens and plants related to such predisposition remains unclear. Here, we investigated the predisposition of Carya cathayensis to infection by the fungal pathogen Botryosphaeria dothidea (Bd) caused by drought in the host plant. High levels of drought stress resulted in a significant increase in plant susceptibility to Bd. Drought significantly induced the accumulation of H2O2 and the free amino acids Pro, Leu, and Ile, and in the phloem tissues of plants, and decreased the content of non-structural carbohydrates. In vitro assays showed that Bd was sensitive to H2O2; however, Pro played a protective role against exogenous H2O2. Leu, Ile, and Pro induced asexual reproduction of Bd. Our results provide the first analysis of how drought predisposes C. cathayensis to Botrysphaeria canker via amino acid accumulation in the host plant, and we propose a model that integrates the plant-pathogen interactions involved.

Cytotoxic, antioxidant, and cytoprotective properties of polyphenol-enriched extracts from pecan nutshells in MDA-MB-231 breast cancer cells.

Phenolic compounds present in plants have demonstrated several biological properties such as antioxidant, antitumor, cardioprotective, and antiproliferative. On the other hand, doxorubicin, a chemotherapeutic widely used to treat breast cancer, usually exhibits chronic cardiotoxicity associated with oxidative stress. Therefore, we aimed to study the effects of phenolic compound-enriched extract (PCEE) with doxorubicin in breast cancer. To achieve this, after an SPE-C18 -column purification process of crude extracts obtained from pecan nutshells (Carya illinoinensis), the resulting PCEE was used to evaluate the cytotoxicity and antioxidant properties against the human breast cancer cell line MDA-MB-231 and the normal-hamster ovary cell line CHO-K1. PCEE was selectively cytotoxic against both cell lines, with an IC50 value (≈26.34 mg/L) for MDA-MB-231 lower than that obtained for CHO-K1 (≈55.63 mg/L). As a cytotoxic mechanism, PCEE inhibited cell growth by G2/M cell cycle arrest in MDA-MB-231 cells. Simultaneously, the study of the antioxidant activity showed that PCEE had a cytoprotective effect, evidenced by reduced ROS production in cells with oxidative stress caused by doxorubicin. The results highlight PCEE as a potential antitumor agent, thus revaluing it as an agro-industrial residue.

First Report of Colletotrichum aenigma causing Anthracnose on Pecan in China.

Pecan (Carya illinoinensis) is an important economic forest crops widely cultivated in China. From June to September in both 2021 and 2022, severe leaf disease resembling anthracnose was observed in 6.6-ha pecan orchard in Jintan (31°42'23.84″ N, 119°21'22.90″ E), Jiangsu Province. The disease severity was about 15 to 25% with 5 to 12% incidence on 100 surveyed trees of the orchard in 2022. Symptoms initially appeared as small gray-bark sunken lesions, which gradually developed to big sunken lesions with brown edges and irregular-shaped. Small fragments (4 × 4 mm) from the necrotic borders of infected leaves were surfaced sterilized, plated on potato dextrose agar (PDA) and then incubated in darkness at 25°C for 3 days. Pure cultures were obtained by monosporic isolation. Twenty-one isolates with similar characteristics were obtained from the infected leaves (isolation frequency about 90%). The upper side of colonies on the PDA plates was milky, and the reverse side was pale yellow at the center and pale white at the margin. After 10 days of growth on the PDA medium, these isolates produced spores separately. . Through electron microscopic observation, conidia were smooth walled, hyaline, aseptate, guttulate, cylindrical with rounded ends with 15 to 20.5 × 5.3 to 6.7 µm (mean 18.5 × 5.8 µm, n = 50) in size. These morphological characteristics were similar to those of the species of Colletotrichumspp (Weir et al. 2012, Fu et al. 2019). To further identify the isolates, the regions of internal transcribed spacer (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHSI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin 2 (TUB2) loci of the three representative isolates (JSJT-1, JSJT-2, and JSJT-3) were amplified and sequenced with the primer pairs ITS-1F/ITS-4, ACT-512F/ACT-783R, CL1/CL2A, CHS-79F/CHS-345R, GDF/GDR and T1/T2 primers, respectively (Weir et al. 2012). Sequences of them were deposited in GenBank under nos. OR214960 to OR214962 (ITS), OR228543 to OR228545 (ACT)，OR228546 to OR228548 (CAL), OR228549 to OR228551 (CHSI), OR228552 to OR228554 (GAPDH), and OR228555 to OR228557 (TUB2). Multilocus phylogenetic analysis revealed that the three isolates and C. aenigma were clustered in the same clade. Based on the results of morphological and molecular analysis, these isolates were identified as C. aenigma. The pathogenicity of three isolates was tested on leaves of pecan seedlings. Suspensions of conidia were obtained by scraping the surface of a 10-day-old sporulated petri dish PDA cultures into sterile water. Suspensions were adjusted to a density of 2 × 106 conidia/ml with a hemocytometer.The conidial suspension of each isolate was sprayed evenly on the surface of leaves from three healthy pecan seedlings. Sterilized distilled water was used for negative controls. The pathogenicity experiment was repeated three times. Finally, all inoculated plants were kept in a light-incubator at 28°C under 100% relative humidity and 12 h photoperiod. Two weeks after inoculation, the inoculated plants developed symptoms similar to those of the original diseased plants, while controls remained asymptomatic. C. aenigma were re-isolated from from inoculated leaves. C. aenigma has been reported as the causal agent of anthracnose on several economically important plants, such as grape ( Kim et al. 2021), tree peonies (Wang et al.2023), chili (Diao et al. 2017), and pear (Fu et al. 2019), but this is the first report of C. aenigma causing anthracnose on pecan in China. Identification of C. aenigma as a pathogen of pecan is important for implementing control management strategies for pecan disease. References: Diao, Y. Z., et al. 2017. Persoonia. 38:20. Fu, M., et al. 2019. Persoonia. 42:1. Kim, J. S., et al. 2021. Plant Dis. 105:2729. Weir, B. S., et al. 2012. Stud. Mycol.. 73:115. Wang, Y. L., et al. 2023. Plant Dis. 107(4):1242. The author(s) declare no conflict of interest. Keywords: Colletotrichum aenigma, Anthracnose, Carya illinoinensis, Pathogenicity.

First report of Colletotrichum fructicola causing anthracnose on Carya cathayensis Sarg. in China.

Carya cathayensis Sarg. (Chinese hickory) is one of the important economic forest plants, mainly distributed in Zhejiang and Anhui provinces in China. In September 2020, leaf spot disease occurred on 90% C. cathayensis in a 2.6 km2 plantation with 500 hickorys in Shangshu Village (30°26'N, 119°32'E), Huzhou, Zhejiang, China. Symptoms initially appeared as small brown spots. Later, the spots became dark brown, and joined into irregular shapes. Twenty diseased leaves with typical symptoms were collected and used to isolate the pathogen. The leaf tissues (5 × 5 mm) at junction of diseased and healthy portion were cut and surface-sterilized with 75% ethanol for 15 s, 0.1% NaClO for 2 min, and rinsed 3 times in sterile water, then placed on potato dextrose agar (PDA) plates and incubated at 25°C in the darkness for 3 days. Eight isolates with similar morphological characterizations were obtained after pure cultures by transferring hyphal tips. The colony growing on PDA for 7 days was circular, dense, white cotton-like hyphae, and light gray-black hyphae can be seen inside. The conidia were cylindrical, aseptate, hyaline, with rounded ends, and 12.5 to 20.0 × 5.0 to 7.5 µm (n = 50). The appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and were in the range of 6.4 to 10.2 × 5.0 to 6.7 µm (n = 50). The morphologies of the isolates were consistent with the genus description of Colletotrichum (Fuentes-Aragón et al. 2018; Liu et al. 2015). The internal transcribed spacer (ITS) regions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), chitin synthase (CHS-1), beta-tubulin 2 (TUB2) and actin (ACT) genes were amplified from genomic DNA for the isolates using the primers described by Weir et al. (2012). The sequences of eight isolates were consistent and the representative isolate CFZJ-64 were deposited in GenBank under the following accession numbers: ITS, OK145563; ACT, OK216738; CAL, OK216739; CHS-1, OK216740; GAPDH, OK216741; and TUB2, OK216742. A phylogenetic tree was generated by combining ITS, ACT, CAL, CHS-1, TUB2, and GAPDH sequences in MEGA11. Three representative isolates CFZJ-42, CFZJ-53 and CFZJ-64 clustered in the C. fructicola clade with 90% bootstrap support. Based on morphological characteristics and phylogenetic analysis, the isolates were identified as C. fructicola. To confirm pathogenicity, 9 detached healthy leaves and 9 healthy leaves on 3-year-old C. cathayensis seedlings were inoculated with conidial suspension of each isolate (20 µL, 1 × 106 conidia/mL). The control leaves were treated with distilled water (20 µL). Each tested leaf was covered with a clean ziplock bag and incubated for 48h at about 27°C, and 14h photoperiod. After five days, 7 of 8 isolates caused on all detached leaves or part of the leaves on the seedlings developed lesions similar to those observed in the field, whereas controls were asymptomatic. The same fungus was re-isolated from all the diseased leaves and identified by sequencing, confirming Koch's postulates. As far as we know, this is the first report of C. fructicola causing anthracnose on C. cathayensis. This study not only expands the knowledge on this important pathogen of C. cathayensis in China, but also provides the foundation to further investigate the biology, epidemiology, and control of the disease.

First Report of Diaporthe pseudophoenicicola causing Leaf Blight on Pecan in China.

Pecan (Carya illinoinensis) is one of the important economic forest crops widely cultivated in Jiangsu Provinces, China. From August to September in both 2021 and 2022, a foliar blight was observed in 7-ha and 6-ha pecan orchards in Changzhou (31°58'9.6″ N, 119°48'33.84″ E), and Jurong (31°52'15.46″ N, 119°9'24.62″ E), Jiangsu Province. The disease severity was about 32% with 8% incidence on 120 surveyed trees of the two orchards. Typical symptoms were lesions with a dark-brown color, which later became brown. We collected eighteen pecan leaves with typical symptoms in the surveyed pecan orchards and took them back to the laboratory for identification. Small fragments (approximately 9 mm2) from the necrotic borders of infected leaves were surfaced sterilized, plated on potato dextrose agar (PDA) and then incubated in darkness at 25°C. Pure cultures were obtained by single-spore culture. Thirty-three isolates with similar characteristics were obtained from the infected leaves (isolation frequency 85%), and the colonies surface on PDA was ochreous with patchs of olivaceous-yellow and sparse aerial mycelium. Observing from the back of the plate, the colonies were cream-yellow. Two types of single-cell conidia were produced on PDA. Alpha-conidia were 7.4 (range, 5.9 to 8.8) × 2.1 (range, 1.6 to 2.8) µm (n = 100), aseptate, smooth, fusiform, straight and tapering towards both ends. Beta-conidia were 25.1 (range, 19.1 to 36.2) × 1.3 (range, 1.0 to 2) µm (n = 100), filiform, hyaline, aseptate and curved at one end. The morphological features of these isolates agreed with those of Diaporthe sp. (Gomes et al. 2013; Gao et al. 2017). To further identify the isolates, the regions of internal transcribed spacer (ITS, OR214967 to OR214969), calmodulin (CAL, OR228558 to OR228560), translation elongation factor 1-α (EF1a, OR228561 to OR228563), histone H3 (HIS, OR228564 to OR228566), and beta-tubulin 2 (TUB2, OR228567 to OR228569) were amplified and sequenced from genomic DNA for the three representative isolates (LSM1, LSM2 and LSM3), respectively (Gomes et al. 2013). Multilocus phylogenetic analysis revealed that the three isolates and D. pseudophoenicicola were clustered in the same clade. Based on the results of morphological and molecular analysis, these isolates were identified as D. pseudophoenicicola. The pathogenicity of three isolates were tested on leaves of pecan seedlings. The conidial suspension (1 × 105 conidia/ml) of each isolate was sprayed evenly on the surface of leaves of three healthy seedlings. Sterilized distilled water was used for negative controls. Finally, all inoculated plants were kept in a greenhouse at 28°C under 100% relative humidity. Two weeks after inoculation, the inoculated plants developed symptoms similar to those of the original diseased plants, while controls remained asymptomatic. D. pseudophoenicicola were re-isolated from from inoculated plants. The pathogenicity experiment was repeated three times. Previously, D. pseudophoenicicola has been reported to cause stem-end browning disease in ripe mango (Takushi et al. 2016; Xu et al 2022). To our knowledge, this is the first report of D. pseudophoenicicola causing leaf blight on pecan . This study provides important information for developing effective pecan disease management practices.

First report of leaf spot on pecan (Carya illinoinensis) caused by Stemphylium eturmiunum in China.

Pecan [Carya illinoinensis (Wangenh.) K. Koch] is an important nut tree species, which has been widely planted in Jiangsu and Anhui Provinces of China in recent years (Mo et al. 2018). In May 2022, a new leaf spot disease was observed on both young and old leaves of pecan trees in the Luhe area, Nanjing, Jiangsu Province. Approximately 30% of pecan trees suffered from the disease, which affected the growth of young trees and nut production to cause economic loss. Initially, the leaf spots were grayish black and round. Then, disease spots enlarged and joined together, forming irregular lesions with uneven edges. In the last stage, the leaflets were withered. To isolate the pathogen, three symptomatic leaves were collected from each of three different pecan trees. Leaf sections (3 to 4 mm) were excised from the margin of spots, surface sterilized in 75% alcohol for 30 s, then sterilized in 1.5% NaClO for 90 s. After rinsing three times with sterile distilled water, leaf sections were placed on potato dextrose agar (PDA) medium and incubated at 25 °C in a dark environment for 5 days. Pure cultures were obtained by monosporic isolation. A total of 20 isolates were obtained, and 12 isolates were identified as Stemphylium sp. with the same morphological features and ITS sequences. A representative isolate, named LH3-3, was selected for further study. Colonies on PDA were light yellow with dense mycelium and were brownish yellow on the reverse side. Conidia were 16.3 to 34.4 × 8.1 to 16.3 µm) (n=35), muriform, brown, with transverse and longitudinal septa, lightly deformed at the transverse septa. Ascomata were not observed. The morphological characteristics were consistent with the description of Stemphylium eturmiunum (Simmons 2001). The internal transcribed spacer region (ITS) and portions of genes for calmodulin (cmdA) and glyceraldehyde-3-phosphate dehydrogenase (gpd) were amplified and sequenced with the primers ITS1/ITS4 (White et al. 1990), CALDF1/CALDR2 (Xu et al. 2022) and GPD-F/R (Xie et al. 2019), respectively. Sequences were deposited in GenBank under accessions OP482492 (ITS), OP495734 (cmdA), and OP495735 (gpd). BLAST analysis showed that the sequences had 99.67-100% homology to ITS (525/525 bp) of S. eturmiunum strain ST14 (MH843733), cmdA (694/694 bp) of strain CBS122124 (KU850832), and gpd (299/300 bp) of isolate UMSe0030 (MK336876). MEGA 7.0 was used to construct a phylogenetic analysis based on concatenated sequences of ITS, cmdA, and gpd using the neighbor-joining method. The results showed that LH3-3 clustered on the branch of S. eturmiunum, and the support rate was 100%. A spore suspension in sterile water was made from strain LH3-3 grown on PDA, and adjusted to 1×106 spores/mL with a hemocytometer. To test pathogenicity, 20 µl drops of the spore suspension were placed on the left sides of four healthy detached leaflets of mature pecan trees and leaves of three 3-month-old seedlings. The right side of each leaflet was inoculated with 20 µl drops of sterile distilled water as the control. All inoculated seedlings and detached leaflets were covered with a transparent plastic bag and cultured in a greenhouse at 25 °C, 80% relative humidity, and a 12 h light cycle until symptom appeared. The experiment was repeated three times. After 7 days of inoculation, grayish black lesions appeared on all inoculation sites with the spore suspension but not in the controls. The leaf spot symptoms were similar to those observed in orchards. The same fungus, identified by morphological characteristics and sequencing of ITS, cmdA, and gpd, was re-isolated from the diseased spots of the inoculated leaflets to complete Koch's postulates. S. eturmiunum has been reported to infect garlic (Dumin et al. 2022) and tomato (Prencipee et al. 2021), but this is the first report of S. eturmiunum causing leaf spot of C. illinoinensis. This study provides a basis for further study on the biology, epidemiology, and management of the disease.

Insight into the CBL and CIPK gene families in pecan (Carya illinoinensis): identification, evolution and expression patterns in drought response.

BACKGROUND: Calcium (Ca2+) serves as a ubiquitous second messenger and plays a pivotal role in signal transduction. Calcineurin B-like proteins (CBLs) are plant-specific Ca2+ sensors that interact with CBL-interacting protein kinases (CIPKs) to transmit Ca2+ signals. CBL-CIPK complexes have been reported to play pivotal roles in plant development and response to drought stress; however, limited information is available about the CBL and CIPK genes in pecan, an important nut crop. RESULTS: In the present study, a total of 9 CBL and 30 CIPK genes were identified from the pecan genome and divided into four and five clades based on phylogeny, respectively. Gene structure and distribution of conserved sequence motif analysis suggested that family members in the same clade commonly exhibited similar exon-intron structures and motif compositions. The segmental duplication events contributed largely to the expansion of pecan CBL and CIPK gene families, and Ka/Ks values revealed that all of them experienced strong negative selection. Phylogenetic analysis of CIPK proteins from 14 plant species revealed that CIPKs in the intron-poor clade originated in seed plants. Tissue-specific expression profiles of CiCBLs and CiCIPKs were analysed, presenting functional diversity. Expression profiles derived from RNA-Seq revealed distinct expression patterns of CiCBLs and CiCIPKs under drought treatment in pecan. Moreover, coexpression network analysis helped to elucidate the relationships between these genes and identify potential candidates for the regulation of drought response, which were verified by qRT-PCR analysis. CONCLUSIONS: The characterization and analysis of CBL and CIPK genes in pecan genome could provide a basis for further functional analysis of CiCBLs and CiCIPKs in the drought stress response of pecan.

A comparative study of antimicrobial, anti-quorum sensing, anti-biofilm, anti-swarming, and antioxidant activities in flower extracts of pecan (Carya illinoinensis) and chestnut (Castanea sativa).

Antibiotic resistance, which has increased rapidly in recent years because of uncontrolled and unconscious antibiotic consumption, poses a major threat to public health. The inadequacy of existing antibiotics has increased the need for new, effective, and less toxic antibiotic raw materials or antibiotic derivatives. Pecan (Carya illinoinensis) and Chestnut (Castanea sativa) flowers possess abundant pollen contents and exhibit similar morphological features. The purpose of this study was to compare these two flower extracts in terms of their antimicrobial and antioxidant activities. Total phenolic content, total flavonoid contents, and phenolic components were also analyzed in aquatic and ethanolic extracts. Antioxidant activities were measured using ferric reducing/antioxidant capacity (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. Antimicrobial and antifungal activities were compared by means of agar diffusion tests against bacteria including Staphylococcus aureus, Bacillus cereus, Mycobacterium smegmatis, Acinetobacter haemolyticus, and Chromobacterium violaceum, and the yeasts Candida albicans and Candida parapsilosis. Anti-quorum sensing (anti-QS), anti-biofilm, and anti-swarming (SW) activities were also studied against Chromobacterium violaceum ATCC 31532, Chromobacterium violaceum ATCC 12472, and Pseudomonas aeruginosa PA01, respectively. Both extracts were rich in ellagic acid and gallic acid and exhibited similar antioxidant properties. Both flower extracts exhibited high antimicrobial and antifungal activities as well as anti-biofilm, anti-QS, and anti-SW activities.

First Report of Bacterial Canker of Pecan Caused by Pseudomonas syringae pv. syringae in China.

Pecan (Carya illinoinensis) is a world-famous nut tree that is widely cultivated in China, especially in Jiangsu Province (Zhang et al. 2015). In April 2022, cankers on trunks were recorded in pecan (cv. Pawnee) fields located in Taizhou (32°27'58″ N, 120°0'49″ E), Jiangsu. Cankers on the trunks resulted in wilt of the plants. Usually, the color of infected bark on the trunk became darker than the healty bark. When the outer bark was peeled away, the inner tissues were water-soaked, often with reddish streaks. In the surveyed orchards, disease incidence ranged from 10 to 20% among young saplings (about 200 three-year-old trees). While no fungal mycelium or spores were found in the diseased areas by microscope, bacterial colonies were isolated by surface-sterilizing small fragments (25 mm2) of symptomatic tissue in 0.5% NaOCl, rinsing the sections twice in sterilized water, and then streaking them on Luria-Bertani (LB) plates. More than 20 bacterial isolates were obtained and all isolates induced a hypersensitive response on Nicotiana tabacum. All isolates were fluorescent on King's medium B, and were gram-negative based on lysis by KOH. Isolates were positive for levan formation, negative for oxidase and arginine dihydrolase, and did not cause soft rot on potato slices. Based on above information, the isolates thus belonged to Lelliot's LOPAT group 1, P. syringae (Lelliott and Stead 1988). The 16S rRNA sequences of five representative isolates (accession numbers OP175939-OP175943) were amplified by PCR, sequenced, and compared with the NCBI GenBank database (Weisburg et al. 1991; Sarkar and Guttman 2004), finding a 99.92% genetic similarity with a previously reported 16S rRNA sequence of a Pseudomonas syringae pv. syringae (Pss) isolate (accession numbers NW389777). Additional housekeeping genes gap1(accession numbers OP186937-OP186941), rpoD (accession numbers OP186952-OP186956), gyrB (accession numbers OP186947-OP186951), and gltA (accession numbers OP186942-OP186946) were PCR-amplified and sequenced as reported by Hwang et al. (2005), followed by multilocus sequence typing analysis (MLSA). Molecular phylogenetic trees (MEGA vesion 6.0, maximum likelihood with Jukes-Cantor model, 1,000 bootstraps) were generated based on each of these five DNA regions and revealed that all five isolates were clustered together with the strains in P. syringae genomospecies 2, and grouped these isolates with Pss in the PAMDB database (Hwang et al. 2005). As a result, these isolates were identified as Pss. Pathogenicity on pecan (cv. Pawnee) was confirmed by cutting the trunks of two-year-old pecan trees with sterilized blades dipped in cell suspensions containing 107 CFU/ml of each isolate. Plants inoculated in a similar manner with sterile water served as negative controls. The inoculated plants were incubated in a greenhouse maintained at 25°C and 80% relative humidity. After 7 to 8 days, all inoculated plants showed the symptoms of necrosis previously described for the original field plants, while the control plants did not show symptoms. The bacteria reisolated from the inoculated plants were identified as Pss using the LOPAT tests. These results and the sequence analysis of the 16S rRNA and four housekeeping genes described above, fulfilled Koch's postulates. No target bacteria were isolated from the control plants. To our knowledge, this is the first report of Pseudomonas syringae pv. syringaecausing bacterial canker of pecan worldwide. The identification of this pathogen will allow the study of strategies for managing the disease. References: Hwang, M. S., et al. 2005. Applied and Environmental Microbiology, 71:5182-5191. Lelliott, R. A., and Stead, D. E. 1988. Blackwell Scientific, Sussex, UK. Sarkar, S. F., and Guttman, D. S. 2004. Applied and Environmental Microbiology, 70:1999. Weisburg, W. G., et al. 1991. Journal of Bacteriology, 173: 697. Zhang, R., et al. 2015. Scientia Horticulturae, 197: 719-727. The author(s) declare no conflict of interest. Keywords: Carya illinoinensis, Pseudomonas syringae, Canker, Identification †Indicates the corresponding author.Y. Q. Zhao; zhaoyuqiang123@126.com.

First Report of Colletotrichum siamense causing Anthracnose on Pecan in China.

Pecan (Carya illinoinensis) is one of the important economic forest crops which has been widely cultivated in Anhui and Jiangsu Provinces, China. Since 2019, symptoms resembling anthracnose disease had been observed in 5-ha and 6.6-ha pecan orchards in Quanjiao ( 32°5'7.08″ N, 118°16'2.91″ E), Anhui Province, and Jintan (31°42'23.84″ N, 119°21'22.90″ E), Jiangsu Province. The disease severity was about 20 to 30% with 5 to 15% (about 500 trees) incidence. In May, symptoms of leaf initially appeared as small dark lesions, which gradually developed to irregular-shaped, sunken lesions (Figure S1, A). From August to October, similar symptoms were also observed on the fruits. Infected fruits appeared irregularly, dark and depressed necrotic lesions on which orange spore masses could be occasionally observed (Figure S1, B). As the disease progressed, the necrotic lesions gradually expanded and merged, resulting in abscission of the fruits. Small fragments (4 × 4 mm) from the necrotic borders of infected fruits or leaves were surfaced sterilized, plated on potato dextrose agar (PDA) and then incubated in darkness at 25°C for 3 days. Pure cultures were obtained from individual conidia by recovering single spores. On the PDA plate, the colonies surface was white and cottony. Observing from the back of the plate, the colonies were pale yellow at the centre and pale white at the margin (Figure S1, E). Spores were produced over PDA plates after 7 days growth. Conidia were hyaline, smooth walls, aseptate, guttulate, cylindrical with rounded ends with 14.8 to 17.5 × 3.3 to 4.7 µm (mean 16.5 × 4.1µm, n = 50) in size (Figure S1, F). These morphological characteristics were similar to those of the species of Colletotrichum siamense (Prihastuti et al. 2009; Weir et al. 2012; Fu et al. 2019). Thirty-two isolates Colletotrichum sp. were obtained from the infected leaves and fruits (isolation frequency about 80%). To further identify the isolates, the regions of internal transcribed spacer (ITS), calmodulin (CAL), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHSI), and beta-tubulin 2 (TUB2) were amplified and sequenced from genomic DNA for the four representative isolates (JS1 and AH1 from infected fruits; JS2 and AH2 from infected leaves), respectively (Weir et al. 2012). Sequences of them were deposited in GenBank under nos. OP389224 to OP389227 (ITS), OP413765 to OP413768 (CAL), OP413761 to OP413764 (ACT), OP413773 to OP413776 (GAPDH), OP413769 to OP413772 (CHSI), and OP413777 to OP413780 (TUB2). Blast analysis showed these sequences shared high identity with C. siamense (100% with ITS, CAL, CHSI, and TUB2; 98.94% with ACT; 98.19% with GAPDH). Multilocus phylogenetic analysis revealed that the four isolates and C. siamense were clustered in the same clade (Figure S2). Based on the results of morphological and molecular analysis, these isolates were identified as C. siamense. The pathogenicity of four isolates was tested on two-year-old container-grown pecan seedlings, which were grown in the nursery. The conidial suspension with a concentration of 5 × 106 conidia/ml was sprayed evenly on the surface of leaves of a healthy seedling, and each isolate inoculated three pecan seedlings. The pathogenicity experiment was repeated three times. For negative controls, pecan seedlings were sprayed with sterilized distilled water. Finally, all inoculated plants were kept in a greenhouse at 25°C under a 16 h/8 h photoperiod and 70% relative humidity. Three weeks after inoculation, the inoculated plants showed symptoms similar to those of the original diseased plants (Figure S1, C), while controls remained asymptomatic (Figure S1, D). Cultures were re-isolated from the infected leaves and were identified as C. siamense by both morphological characteristics and DNA sequence analysis. Previously, C. nymphaeae, C. siamense, C. fructicola and C. viniferum have been reported to cause anthracnose of Pecan worldwide (Zhang et al. 2019; Oh et al. 2021; Poletto et al. 2019; Zhao et al. 2022 ). To our knowledge, this is the first report of C. siamense causing anthracnose on pecan in China. The identification of this pathogen will facilitate the development of strategies for managing the disease in China. References: Oh, J. Y., et al. 2021. Plant disease. 105(10):3296. Poletto, T., et al. 2019. Plant disease. 103(12):3277. Prihastuti, H., et al. 2009. Fungal Divers. 39:89. Fu, M., et al. 2019. Persoonia-Molecular Phylogeny and Evolution of Fungi. 42(1):1-35. Weir, B. S., et al. 2012. Studies in Mycology. 73:115. Zhao, et al. 2022, Acta Phytopathologica Sinica, doi:10.13926/j.cnki.apps.000648 Zhang, Y. B., et al. 2019. Plant disease. 103(6):1432. The author(s) declare no conflict of interest. Keywords: Colletotrichum siamense, Anthracnose, Carya illinoinensis, Pathogenicity †Indicates the corresponding author. Y. Q. Zhao; zhaoyuqiang123@126.com.

Genome-Wide Identification, Characterization, and Expression Profiling of AP2/ERF Superfamily Genes under Different Development and Abiotic Stress Conditions in Pecan (Carya illinoinensis).

The ethylene-responsive element (AP2/ERF) is one of the keys and conserved transcription factors (TFs) in plants that play a vital role in regulating plant growth, development, and stress response. A total of 202 AP2/ERF genes were identified from the pecan genome and renamed according to the chromosomal distribution of the CiAP2/ERF genes. They were divided into four subfamilies according to the domain and phylogenetic analysis, including 26 AP2, 168 ERF, six RAV, and two Soloist gene family members. These genes were distributed randomly across the 16 chromosomes, and we found 19 tandem and 146 segmental duplications which arose from ancient duplication events. The gene structure and conserved motif analysis demonstrated the conserved nature of intron/exon organization and motifs among the AP2/ERF genes. Several cis-regulatory elements, which were related to light responsiveness, stress, and defense responses, were identified in the promoter regions of AP2/ERFs. The expression profiling of 202 CiAP2/ERF genes was assessed by using RNA-Seq data and qRT-PCR during development (pistillate flowering development, graft union development, and kernel development) and under abiotic stresses (waterlogging, drought). Moreover, the results suggested that the ERF-VII members may play a critical role in waterlogging stress. These findings provided new insights into AP2/ERF gene evolution and divergence in pecan and can be considered a valuable resource for further functional validation, as well as for utilization in a stress-resistance-variety development program.

CcMYB12 Positively Regulates Flavonoid Accumulation during Fruit Development in Carya cathayensis and Has a Role in Abiotic Stress Responses.

Flavonoid, an important secondary metabolite in plants, is involved in many biological processes. Its synthesis originates from the phenylpropane metabolic pathway, and it is catalyzed by a series of enzymes. The flavonoid biosynthetic pathway is regulated by many transcription factors, among which MYB transcription factors are thought to be key regulators. Hickory (Carya cathayensis) is an economic forest tree species belonging to the Juglandaceae family, and its fruit is rich in flavonoids. The transcriptome of exocarp and seed of hickory has previously been sequenced and analyzed by our team, revealing that CcMYB12 (CCA0691S0036) may be an important regulator of flavonoid synthesis. However, the specific regulatory role of CcMYB12 in hickory has not been clarified. Through a genome-wide analysis, a total of 153 R2R3-MYB genes were identified in hickory, classified into 23 subclasses, of which CcMYB12 was located in Subclass 7. The R2R3-MYBs showed a differential expression with the development of hickory exocarp and seed, indicating that these genes may regulate fruit development and metabolite accumulation. The phylogenetic analysis showed that CcMYB12 is a flavonol regulator, and its expression trend is the same as or opposite to that of flavonol synthesis-related genes. Moreover, CcMYB12 was found to be localized in the nucleus and have self-activation ability. The dual-luciferase reporter assay demonstrated that CcMYB12 strongly bonded to and activated the promoters of CcC4H, CcCHS, CcCHI, and CcF3H, which are key genes of the flavonoid synthesis pathway. Overexpression of CcMYB12 in Arabidopsis thaliana could increase the content of total flavonoids and the expression of related genes, including PAL, C4H, CHS, F3H, F3'H, ANS, and DFR, in the flavonoid synthesis pathway. These results reveal that CcMYB12 may directly regulate the expression of flavonoid-related genes and promote flavonoid synthesis in hickory fruit. Notably, the expression level of CcMYB12 in hickory seedlings was significantly boosted under NaCl and PEG treatments, while it was significantly downregulated under acid stress, suggesting that CcMYB12 may participate in the response to abiotic stresses. The results could provide a basis for further elucidating the regulation network of flavonoid biosynthesis and lay a foundation for developing new varieties of hickory with high flavonoid content.

Diversity of Botryosphaeriaceae Species Associated with Chinese Hickory Tree (Carya cathayensis) Trunk Cankers.

Tree trunk cankers represent serious fungal diseases that pose significant threats to Chinese hickory trees (Carya cathayensis). To characterize the pathogen diversity associated with this disease, tissues were collected between 2016 and 2018 from the primary Chinese hickory plantation regions. A total of 97 cultures were isolated from trees in six towns (Longgang, Qingliangfeng, Changhua, Tuankou, Taiyang Town, and Lin'an urban area) within the Linan district, where 60% of Chinese hickory tree yields originate. The isolated cultures caused cankers on Chinese hickory tree branches, but infections did not occur on fruits or leaves under tested conditions. Combined morphological observations and phylogenetic analysis of multiple genes (ITS, ß-tubulin, and EF) indicated that five Botryosphaeriaceae species were recovered, including 89 isolates of Botryosphaeria dothidea, 4 isolates of Botryosphaeriaceae fabicerciana, 1 isolate of Botryosphaeriaceae qingyuanensis, 1 isolate of Botryosphaeriaceae corticis, and two isolates of Lasiodiplodia theobromae. B. dothidea was the most prevalent, and this is the first report of B. corticis, B. qingyuanensis, and L. theobromae infections in Chinese hickory trees. We investigated the mycelial growth, spore germination, and pathogenicity of these species at different temperatures. L. theobromae grew the fastest and B. cortices grew the slowest on potato dextrose agar. The optimum temperature of spore germination for all species was 30°C. L. theobromae was the most virulent species, followed by B. dothidea and B. qingyuanensis, then B. fabicerciana, and finally B. cortices. These new insights into fungal pathogen diversity provide critical new information to understand and manage tree trunk cankers of Chinese hickory.

Effect of Tractor Speed and Spray Application Volume on Severity of Scab and Fruit Weight at Different Heights in the Canopy of Tall Pecan Trees.

Scab (caused by Venturia effusa) is the most important yield-limiting disease of pecan in the southeastern USA. On susceptible cultivars, the disease is managed using fungicides, but spray coverage is an issue in tall trees. In four experiments, we used an air-blast sprayer to compare scab severity on fruit at 5.0 to 15.0 m height in trees receiving the same dose of fungicide at 468, 935, and 1,871 liter/ha at 2.4 and 3.2 km/h (in two additional experiments fungicides were applied at 4.0 km/h at 470 liter/ha, 4.0 km/h at 940 liter/ha and 4.0 km/h at 1,100 liter/ha). An air-blast sprayer was used for the applications, which included typical recommended active ingredients (a.i.). Nozzles were selected to provide similar proportions of spray to the upper and lower canopy. The treatments (or subsets thereof) were repeated in 2015 to 2017 on cv. Schley and in 2017, 2019, and 2020 on cv. Desirable. All treatments reduced scab compared with the control. Overall, there was no consistent difference among the treatments for severity of scab on foliage, immature fruit, or mature fruit at any height in the canopy up to 15.0 m (maximum height sampled). Fungicide applied at 2.4 or 3.2 km/h at 470 liter/ha was as effective at reducing disease as were the higher volumes (sometimes more so). The scab epidemic severity affected control efficacy. Estimated cost and water savings based on faster speed and lower volume were considerable. These preliminary observations indicate no single volume or speed was consistently superior to control scab; this suggests that, in most seasons, low volumes (higher concentration of a.i.) may be similarly efficacious as high volumes (lower concentration of a.i.) for controlling scab in tall pecan trees and offer greater resource use efficiency.