Histone H3 Gene Research Articles

First Report of Postharvest Fruit Rot on Gannan Navel Orange Caused by Diaporthe unshiuensis in Jiangxi Province, China.

The Gannan navel orange (Citrus sinensis Osbeck cv. Newhall) is one of the most widely planted citrus fruit cultivars in Ganzhou City, Jiangxi Province, China. A Gannan navel orange was harvested from an orchard in Yudu County, Ganzhou City, Jiangxi Province, China, in October 2022 (25.95N, 115.41E). Approximately 5% of the fruit rotted after being stored at room temperature for about two weeks.Infected fruits appear brown and rotted with slightly indented edges. Initially symptoms of infected fruits was small circular, light brown, which the rot expands, slightly water-stained halo circle with slightly indented edges. The surface of 10 infected fruits was sterilized with 75% ethanol, and the lesion edge was cut into 5-mm-diameter pieces, and the pieces were then placed on potato dextrose agar (PDA) and incubated at 25°C for five days. A total of eight morphologically similar isolates were obtained. PDA results showed dense white and fluffy aerial mycelia in the center of colonies with sparser edges. Two types of conidia were produced; the alpha conidia were hyaline, ellipsoidal or clavate, and aseptate, with 2 oil drops, 4.8 to 7.5 × 2.1 to 2.7 μm (n=30). The beta conidia were hyaline, aseptate, filiform, smooth, straight to sinuous, 16.9 to 27.5 × 1.3 to 1.6 μm (n=30). These isolates exhibit morphological characteristics similar to those of the Diaporthe genus. Genomic DNA of two representative isolates (JFRL-03-1130 and JFRL-03-1131) was extracted for further confirmation. The internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α), and histone H3 (HIS3) genes were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, and CYLH3F/H3-1b, respectively (Udayanga et al. 2015). These nucleotide sequences were deposited into the GenBank database with accession numbers OQ691637-OQ691638 (ITS), OQ701022-OQ701023 (TUB), OQ701016-OQ701017 (CAL), OQ701018-OQ701019 (TEF1-α) and OQ701020-OQ701021 (HIS3). The maximum likelihood analyses were performed for the combined ITS, TEF1-a, TUB, HIS3, and CAL data set using Phylosuite V1.2.2 (Zhang et al. 2020). The phylogenetic tree showed that the two isolates clustered with D. unshiuensis in a clade with 100% bootstrap support. Therefore, the fungus was identified as D. unshiuensis based on morphological and molecular characteristics. To evaluate pathogenicity, a sterile scalpel was used to wound 10 surface-sterilized fruits, and a 5-mm-diameter mycelial plug of the isolate JFRL 03-1130, cultured on PDA at 25℃ for 7-days, was put on the wound. Another set of 10 fruits was similarly inoculated with sterile agar plugs as controls. The fruits were cultured at 25°C and 85% relative humidity, and the test was repeated twice. These fruits inoculated with D. unshiuensis showed similar rot symptoms after 10 days, while the control group remained symptomless. In order to prove Koch's postulates, the pathogen was re-isolated from the inoculated fruits and confirmed as D. unshiuensis by molecular techniques, but never from the control fruits. Diaporthe unshiuensis has been reported as an endophyte associated with citrus and a pathogen that causes melanose disease in citrus (Chaisiri et al. 2020; Huang et al. 2015). However, to the best of our knowledge, this is the first reported case of D. unshiuensis causing postharvest fruit rot on Citrus sinensis. In the past, D. sojae has also been reported causing postharvest fruit brown rot disease on Citrus sinensis in China (Xiao, et al. 2023); Therefore, managers should pay more attention to postharvest fruit rot disease caused by Diaporthe species and implement storage strategies to control and reduce losses.

A review of current therapeutics targeting the mitochondrial protease ClpP in diffuse midline glioma, H3 K27-altered.

Diffuse midline gliomas (DMGs) are devastating pediatric brain tumors recognized as the leading cause of cancer-related death in children. DMGs are high-grade gliomas (HGGs) diagnosed along the brain's midline. Euchromatin is the hallmark feature of DMG, caused by global hypomethylation of H3K27 either through point mutations in histone H3 genes (H3K27M), or by overexpression of the enhancer of zeste homolog inhibitory protein. In a clinical trial for adults with progressive HGGs, a 22-year-old patient with a thalamic DMG, H3 K27-altered, showed a remarkable clinical and radiological response to dordaviprone (ONC201). This response in an H3 K27-altered HGG patient, coupled with the lack of response of patients harboring wildtype-H3 tumors, has increased the clinical interest in dordaviprone for the treatment of DMG. Additional reports of clinical benefit have emerged, but research defining mechanisms of action (MOA) fall behind dordaviprone's clinical use, with biomarkers of response unresolved. Here, we summarize dordaviprone's safety, interrogate its preclinical MOA identifying the mitochondrial protease "ClpP" as a biomarker of response, and discuss other ClpP agonists, expanding the arsenal of potential weapons in the fight against DMG. Finally, we discuss combination strategies including ClpP agonists, and their immunomodulatory effects suggestive of a role for the tumor microenvironment in DMG patient response.

First report of stem blight caused by Diaporthe eres on highbush blueberry (Vaccinium corymbosum) in Michigan.

The U.S. is the world's leading producer of highbush blueberries (Vaccinium corymbosum L.), and Michigan is ranked in the top five production states (USDA NASS, 2022). In June and July of 2021, 268 blueberry stem blight samples were collected for a pathogen survey across 22 total fields in Van Buren and Ottawa counties in Michigan. Current season stems with symptoms of necrosis and wilting were collected. Stems were cut just below the necrotic area and cross-sections (2-3 mm long) were surface disinfested in 10% bleach for 1 min, rinsed twice in sterile distilled water, and dried on sterile paper towels. Stem cross-sections were plated onto potato dextrose agar (PDA) amended with 100 µg/ml streptomycin sulfate and 50 µg/ml ampicillin. Plates were incubated at 21°C under a 12-h photoperiod for 5-6 days. Outgrowing fungi with morphology similar to Diaporthe spp. were transferred to new PDA plates 2 consecutive times after 7 days of similar incubation to ensure single colony isolation. After 7 days, colonies consisted of white and light brown mycelia that were mostly flat, with some isolates that had partially raised mycelia towards the center of the plate. After 3-4 weeks, colonies turned brown and gray and produced dark brown pycnidia. Aseptate, hyaline, fusiform to ellipsoid, biguttulate alpha conidia measuring 5.4 to 7.6 x 2.6 to 3.7 µm (n = 60) were produced. No beta conidia were observed. In total, 3 isolates, representing 3 different farms (37-95 km apart) and cultivars ('Duke', 'Jersey', and 'Bluecrop'), as well as 2 counties, were identified as Diaporthe through colony morphology (Gomes et al. 2013, Udayanga et al. 2014). Amplification and subsequent Sanger sequencing were performed for the internal transcribed spacer (ITS) region and portions of the translation elongation factor (TEF) 1-α, β-tubulin (TUB), and histone H3 (HIS) genes using primers ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), T1/Bt-2b (Glass and Donaldson 1995), and CYLH3F/H3-1b (Glass and Donaldson 1995), respectively. Representative sequences were deposited in NCBI GenBank (accession no. OQ507870-OQ507872 for ITS, and OQ550272-OQ550278 for TEF, HIS, and TUB). BLASTn results revealed 97-100% identity for all 4 genes across other established D. eres isolates reported in Gomes et al. (2013). For example, JMK047 had 99.8% (577/578 bp), 99.7% (327/328 bp), 100% (701/701 bp), and 100% (439/439 bp) homology with ITS, TEF, TUB, and HIS sequences, respectively, of D. eres CBS 439.82 (accession no. KC343090, KC343816, KC344058, KC343574). Koch's Postulates were fulfilled via pathogenicity tests on 2-year-old potted 'Blueray' plants with 2 isolates. Stems were surface sterilized with 1% bleach then 8-mm long pieces of bark were removed using a sterile razor blade to expose the cambium. Plugs of sterile PDA (negative control) or mycelia from 7-day old cultures on PDA (5-mm diameter) were placed onto the cambium layer and sealed with Parafilm. Six stems on unique plants were inoculated per treatment. Plants were grown in a 20.5°C greenhouse with a 14-hr photoperiod. After 3 weeks, the stems inoculated with D. eres isolates showed similar stem blight symptoms to those observed in the field while control stems remained healthy. Re-isolation and sequencing of the ITS region of 3 replicates per treatment with the protocol described above confirmed symptoms correlated with D. eres isolates. This is the first report of D. eres associated with stem blight of highbush blueberry in Michigan, and the second report in the U.S. (Lombard et al. 2014). Increasing prevalence of D. eres in U.S. blueberries may affect disease management programs. References Carbone, I., and Kohn, L. M. 1999. Mycologia 91:553. 10.1080/00275514.1999.12061051. Glass, N. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61:1323. 10.1128/aem.61.4.1323-1330.1995. Gomes, R. R., et al. 2013. Persoonia 31:1. 10.3767/003158513x666844. Lombard, L., et al. 2014. Phytopathol. Mediterr. 51(2):287. 10.14601/Phytopathol_Mediterr-14034. Udayanga, D., Castlebury, L. A., Rossman, A. Y., Chukeatirote, E., and Hyde, K. D. 2014. Fungal Divers. 67:203-229. 10.1007/s13225-014-0297-2. USDA NASS. 2022. Noncitrus Fruits and Nuts 2021 Summary. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, California, USA.

Wide Distribution and Intraspecies Diversity in the Pathogenicity of Calonectria in Soil from Eucalyptus Plantations in Southern Guangxi of China.

Eucalyptus spp. are extensively cultivated in southern China because of their adaptability and versatile timber production. Calonectria leaf blight caused by Calonectria species is considered a major threat to Eucalyptus trees planted in China. The GuangXi Zhuang Autonomous Region is the provincial region with the largest distribution of Eucalyptus plantations in China. The present study aimed to expound the species diversity and pathogenicity of Calonectria isolates obtained from the soil of Eucalyptus plantations in GuangXi. A total of 188 Calonectria isolates were recovered from the soil located close to Eucalyptus trees, and the isolates were identified based on the DNA sequence comparisons of the four partial regions of the translation elongation factor 1-alpha (tef1), β-tubulin (tub2), calmodulin (cmdA), and histone H3 (his3) genes. The isolates were identified as Calonectria aconidialis (74.5%), C. hongkongensis (21.3%), C. pseudoreteaudii (2.1%), C. kyotensis (1.6%), and C. chinensis (0.5%). The inoculation results indicated that 40 isolates representing five Calonectria species were pathogenic to the three Eucalyptus genotypes. Two inoculated experiments consistently showed that the longest lesions were produced by the isolates of C. aconidialis. Some isolates of C. aconidialis, C. hongkongensis, and C. kyotensis produced significantly longer lesions than the positive controls, but not the isolates of C. pseudoreteaudii or C. chinensis. These results indicated that Calonectria isolated from the soil may pose a threat to Eucalyptus plantations. Some Calonectria isolates of the same species differed significantly in their virulence in the tested Eucalyptus genotypes. The resistance of different Eucalyptus genotypes to Calonectria isolates within the same species was inconsistent. The inoculation results in this study suggested that many Calonectria isolates in each species had different levels of pathogenicity, and many Eucalyptus genotypes need to be tested to select disease-resistant Eucalyptus genetic materials in the future. The results of the present study enhance our knowledge of species diversity and the potential damage caused by Calonectria in the soil of Eucalyptus plantations. Our results also provide new insights into the breeding of disease-resistant Eucalyptus genotypes for controlling Calonectria leaf blight in China in the future.

Molecular and Morphological Assessment of Juvenile and Adult Forms in the Giant Worm Eunice Cf. Aphroditois (Annelida: Eunicidae) and Its Phylogenetic Position in the Family.

Eunice aphroditois (Pallas, 1788) is a large polychaete worm (up to 3 m in length) and the type species of the genus. In Japan, a similar but potentially different species, Eunice cf. aphroditois, is distributed mainly in the rocky shores of the temperate and warm Pacific coasts. Juveniles and adults were suggested to be distinguished by their body color. The juvenile form was previously regarded as distinct species, Eunice flavopicta Izuka, 1912 and Eunice ovalifera Fauvel, 1936, although they are now considered synonymous with E. aphroditois. In this study, we revisited the validity of the present taxonomy based on morphological observations including SEM and microCT, and three molecular markers (cytochrome c oxidase subunit I [COI], 16S rRNA, and histone H3 genes) and investigated the phylogenetic position of E. cf. aphroditois in the family Eunicidae using the combined dataset of three genes (COI + 16S rRNA + 18S rRNA). The adult and juvenile forms were different in body size, color, the distribution of the branchiae and subacicular hooks, and maxillae shape, but not in other characteristics. One individual showed an intermediate body color between the two forms. The adult and juvenile forms shared major haplotypes and the maximum K2P genetic distance of COI was 1.7%, which can be considered within intraspecific variation. In the phylogenetic tree based on the combined gene dataset, E. cf. aphroditois was closely related to Eunice roussaei Quatrefages, 1866 and Eunice cf. violaceomaculata Ehlers, 1887, which are large species from the Mediterranean Sea and the Caribbean Sea, respectively.

First Report of Black Rot on Persimmon Fruit Caused by Diaporthe eres in China.

The fruit of persimmon (Diospyros kaki L.) is popular with consumers for its delicious taste and is widely grown in China. In September 2022, fruit rot symptoms were found on persimmons grown at the Tobacco Research Institute of Chinese Academy of Agricultural Sciences (120°26'43.879″E, 36°7'59.794″N) in Qingdao City, Shandong Province, China. Diseases incidence on fruit is 70%. Typical symptoms were small, black spots, which gradually expanded, and became rot lesions, causing fruit rot and serious losses. To isolate the pathogen, small sections (approximately 2 × 2 mm) of symptomatic tissues from five persimmon fruits were surface-sterilized in 5% NaClO, followed by 75% ethanol, rinsed with sterile distilled water, and then incubated on potato dextrose agar (PDA) plates incubated at 28℃ for 5 days. By single spore isolation method, seven dominant fungal isolates developed with similar colonies. They were initially white and gradually turned to gray after 7 d. The alpha conidia were unicellular, colorless, elliptic or fusiform, and 5.0 to 8.0 μm × 2.5 to 3.5 μm in size. Beta conidia were unicellular, colorless, filiform, often hooked at one end and 18 to 28 μm × 1.3 to 1.5 μm in size. These morphological features were consistent with Diaporthe spp (Udayanga et al. 2014). To confirm identity of the causal fungus, the internal transcribed spacer (ITS) rDNA, translation elongation factor 1-a (TEF1-a), b-tubulin (TUB), and histone H3 (HIS) genes of three representative isolates (De2, De3 and DeR2) were amplified and sequenced using primers ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b and CYLH3F/H3-1b as described by Chaisiri et al. (2021). The resulting sequences were deposited in GenBank (accession nos. OP963195, OP963386 and OP963389 for ITS; OP968958, OP968959 and OP9689600 for EF1-α; OP978141, OP978142 and OP978143 for TUB; OP968955, OP96896 and OP968957 for HIS), and showed 99-100% similarity with D. eres by NCBI blast. Phylogenetic analysis was conducted using MEGA-X software, with the NJ method. In the phylogenetic tree, the combined sequences attributed the three isolates to the D. eres. On the basis of morphological and molecular characteristics, the pathogen was identified as D. eres. The strain De2 was preserved in the Agricultural Culture Collection of China (preservation number: ACCC 35482). Pathogenicity tests were performed by wound inoculation on healthy persimmon fruit. These were surface sterilized with 75% ethanol for 1 min, rinsed in sterile water three times, and allowed to dry. A 5-mm agar disc with mycelium of the causal pathogen was placed on the surface of fruit, which was wounded with a sterile needle in advance, with the sterile PDA plugs used as control. Three persimmons were used for each treatment, repeat three times. All the treated fruits were incubated in the sterilized transparent plastic cans at 28°C and 90% RH. After 3 days inoculation, the first symptom of rot lesion appeared on the fruit with mycelial plugs, whereas the control fruit remained symptomless. The same causal pathogens were re-isolated from the lesions and identified by morphologyand confirmed to fulfill Koch's postulates. This is the first report of D. eres causing fruit rot on persimmon. This finding showed that Diaporthe eres is a causal agent of persimmon fruit rot in China.

First Report of Diaporthe fusicola Causing Postharvest Fruit Rot of Peach (Prunus persica) in China.

In May 2022, rot symptoms were observed on postharvest peach (Prunus persica [L.] Batsch) fruits in a market in Nanchang, Jiangxi province (28°44' N; 115°50' E), China. A total of 80 samples were collected from three different fruit stalls through the market survey. The incidence of this disease was 10 to 15%, and severity varies from approximately 30 to 50% of fruit surface coverage. The symptom of infected fruits was circular, pale brown to brown, rotten, necrotic lesions, covered with white hyphae and small spore masses. Eight symptomatic peach fruits were surface disinfected with 75% ethanol for 30 sec and incisions were made with a sterile scalpel. Small pieces from symptomatic tissues were placed on a potato dextrose agar (PDA) medium and incubated at 25℃ for 7 days. Six isolates were obtained in total. Colonies on PDA were initially white, aerial, fluffy at first, and darkened with age. Alpha conidia were fusoid, hyaline, aseptate, guttulate, tapering towards ends, and ranged in size from 9.8 to 5.1 µm × 3.2 to 2.1 µm (x ̅=7.1 ± 1.0 × 2.6 ± 0.3 µm, n=60). Beta conidia were not seen. For further confirmation, genomic DNA was extracted from three isolates (04-10, 04-11, and 04-12), the internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α) and histone H3 (HIS) genes were amplified by using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, CYLH3F/H3-1b (Udayanga et al. 2015), respectively. Sequences were deposited in GenBank (Accession Nos. ON994257 to ON994259 for ITS, OP076824 to OP076826 for TUB, OP076827 to OP076829 for CAL, OP076821 to OP076823 for TEF1-α, OP076830 to OP076832 for HIS). BLAST results showed that ITS and TEF1-α have 99.8% pairwise identity to Diaporthe fusicola (MN816432, KF576256), and the TUB, CAL, and HIS sequences also have 100% pairwise identity to D. fusicola (KF576287, MT978147, MT978142). Phylogenetic analyses of concatenated sequences using Bayesian inference and the maximum likelihood confirmed the identity. To verify Koch's postulates, the pathogenicity of three isolates was tested on harvested healthy peach fruits. Five surface-sterilized fruits were wounded by a sterile scalpel and inoculated with 5-mm-diameter mycelial plugs from 10-day-old PDA plates. Another set of five fruits was inoculated with sterilized PDA plugs as controls. All fruits were incubated at 26℃ with 80% relative humidity. The experiment was repeated three times. After 5 days, the fruit inoculated with mycelial plugs showed pale brown lesions with whitish mycelium mass, similar to the previous rot symptoms, whereas the control fruit remained symptomless. The same pathogen was reisolated from the inoculated fruit with symptoms and identified as D. fusicola by molecular techniques, but never from the control. Diaporthe fusicola (Diaporthe amygdali complex) was first described on leaves of Lithocarpus glabra in China (Gao et al. 2015) and reported as an agent causing leaf blotch on Osmanthus fragrans (Si et al. 2020) and pear shoot canker (Guo et al. 2020). However, this is the first report of D. fusicola causing postharvest fruit rot on peach. The managers involved must consider the impact of this disease and develop an effective fruit storage strategy.

Molecular Phylogeny and Taxonomy of the Coral Genus Cyphastrea (Cnidaria, Scleractinia, Merulinidae) in Japan, With the First Records of Two Species.

The scleractinian coral genus Cyphastrea is widely distributed in the Indo-Pacific region and is common from the subtropical to the warm-temperate regions in Japan. Three new species in this genus have recently been reported from south-eastern Australia or the Red Sea. However, taxonomic and species diversity have been little studied so far in Japan. In this study, we analyzed 112 specimens of Cyphastrea collected from the subtropical to the warm-temperate regions in Japan to clarify the species diversity in the country. This analysis was based on skeletal morphological and molecular analyses using three genetic markers of the nuclear 28S rDNA, histone H3 gene, and the mitochondrial noncoding intergenic region between COI and tRNAmet. The molecular phylogenetic trees showed that our specimens are separated mainly into four clades. Considering the morphological data with the molecular phylogenetic relationships, we confirmed a total of nine species, including two species, C. magna and C. salae, recorded for the first time in Japan. Although eight out of nine species were genetically included within Cyphastrea, one species, C. agassizi, was genetically distant from all other species and was closely related to the genus Leptastrea, suggesting the return of this species to the genus to which it was originally ascribed. Two newly recorded species were reciprocally monophyletic, while the other six species (excluding C. agassizi) clustered in two clades without forming species-specific lineages, including three polyphyletic species. Thus, the species boundary between species in Cyphastrea remains unclear in most species using these three sequenced loci.

Overexpression of Rice Histone H1 Gene Reduces Tolerance to Cold and Heat Stress.

Temperature stresses, including low- and high-temperature stresses, are the main abiotic stresses affecting rice yield. Due to global climate change, the impact of temperature pressure on rice yield is gradually increasing, which is also a major concern for researchers. In this study, an H1 histone in Oryza sativa (OsHis1.1, LOC_Os04g18090) was cloned, and its role in rice's response to temperature stresses was functionally characterized. The GUS staining analysis of OsHis1.1 promoter-GUS transgenic rice showed that OsHis1.1 was widely expressed in various rice tissues. Transient expression demonstrated that OsHis1.1 was localized in the nucleus. The overexpression of OsHis1.1 reduces the tolerance to temperature stress in rice by inhibiting the expression of genes that are responsive to heat and cold stress. Under stress conditions, the POD activity and chlorophyll and proline contents of OsHis1.1-overexpression rice lines were significantly lower than those of the wild type, while the malondialdehyde content was higher than that of the wild type. Compared with Nip, OsHis1.1-overexpression rice suffered more serious oxidative stress and cell damage under temperature stress. Furthermore, OsHis1.1-overexpression rice showed changes in agronomic traits.

Development and evaluation of an RNase H-dependent PCR (rhPCR) and an rh-quantitative PCR (rhqPCR) system for detection of a new potato pathogen-Ilyonectria pseudodestructans.

Ilyonectria pseudodestructans is a plant pathogen causing root rot on fruit trees such as grapevine and apple. Recently, it was reported to be a pathogen of potato. The increasing risk of this pathogen on plants makes it essential to develop a rapid and accurate detection method. In this study, an RNase H-dependent PCR (rhPCR) protocol and a modified probe based rh-quantitative PCR (rhqPCR) protocol for I. pseudodestructans detection were developed. Both the forward and reverse primers for rhPCR and rhqPCR carry a RNA nucleotide at the site where a single-nucleotide polymorphism between I. pseudodestructans and strains of other Ilyonectria species is located and the rhqPCR also contains a fluorescent-labeled target-specific probe. The primers were designed based on the sequence of the Histone H3 gene and could amplify a DNA fragment of 73 base pairs (bp). In the specificity test, by alignment via the Blastn tool, the RNA nucleotide bases on both the forward and reverse primers were identical to the corresponding genomic site of 16 of 17 (94.1%) database-available I. pseudodestructans strains, and different to 43 of 44 (97.7%) database-available strains of other Ilyonectria species. When the rhPCR/rhqPCR protocols were applied on 11 I. pseudodestructans strains and 46 other strains of different species of plant pathogens, all the I. pseudodestructans generated positive reactions but all the other strains were negative, which indicated an excellent specificity of the primers. In the sensitivity test, the lowest DNA template amount for a positive reaction using the rhPCR/rhqPCR methods was 2 pg for I. pseudodestructans genomic DNA. When testing the rhqPCR method on gBlock, the lowest number of molecules for a positive reaction was six. These results indicated a high sensitivity of the protocol for I. pseudodestructans detection. This is the first report of a probe-based rhqPCR to be applied on plant disease diagnosis; in addition, this is also the first rapid molecular protocol to detect I. pseudodestructans, and therefore, the new rhPCR/rhqPCR methods are recommended to plant disease diagnostic labs for their routine work.

Abstract 4740: Molecular effects of histone deacetylase inhibitor Quisinostat on diffuse midline glioma of the pons

Abstract Diffuse Midline Glioma of the pons (DMG) is a lethal, aggressive heterogeneous brain stem tumor. Median overall survival is less than a year, with radiation as the only standard treatment. Recently, mutations in DMGs have arisen as potential therapeutic targets, specifically a mutation in one of the histone H3 genes, resulting in methionine substituted for lysine at site 27 (H3K27M). H3K27M induces a marked reduction in global acetylation of histone tails, altering chromatin structure and causing aberrant gene expression. Histone deacetylase inhibitors, HDACis, are epigenetic drugs that show anticancer activity. In our studies, Quisinostat (Quis) is used to preserve histone acetylation. Using two H3K27M-DMG treatment-naive preclinical models (PBT22 and PBT29) we detected a 100-fold differential response to the histone deacetylase inhibitor, Quisinostat. PBT-22 harbors mutations in H3F3A, TP53, and ASXL2, while PBT-29 has mutations in H3F3A, TP53, PIK3CA and FGFR1. Following Quis treatment (48 hrs) in both preclinical models, total H3K27ac protein abundance increased 3-fold, suggesting HDACi stabilizes or impedes turnover of K27 acetylated H3 histone. We are pursuing studies to test whether sensitivity to HDACi in DMGs is determined by a shift in relative or total abundance of respective H3wt- and H3K27M- histones. We posit that changes in H3K27ac manifest as shifts in nucleosome integration with genes responsible for cell survival/death. This project will profile differentially expressed genes (DEGs) from Quis-treated PBT22 and PBT29. Additionally, total and relative abundance of H3 proteins (wt and mt, me, me2, me3, and ac) from treated cells will be determined. Gene ontology analysis will focus on pathways accounting for chromatin remodeling, cell death, and growth arrest. RNA (qRT-PCR) and proteins (western blot) from analytes from a larger panel of DMG cell lines and neural stem cells treated with Quis will be used to validate the findings. Overall, the data depicts DMG preclinical models with large differential sensitivity to Quis, which may be partially due to different oncoprints between the models. The markedly different sensitivity of these models enables mechanistic study of the consequences of elevated abundance of histone 3 acetylation. The long term goal is to discover a molecular profile of DMGs indicative of the vulnerability to HDACi. Citation Format: Danyelle Paine, Nanyun Tang, Yue Hao, Matt Biery, Carrie Meyers, Alyssa Noll, Nicholas Vitanza, Michael Berens. Molecular effects of histone deacetylase inhibitor Quisinostat on diffuse midline glioma of the pons. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4740.

First Report of Diaporthe eres and D. unshiuensis causing Leaf Spots on Sapindus mukorossi in China.

Sapindus mukorossi Gaertn., commonly known as soapberry, is widely cultivated as a landscaping tree in Southern China. In June 2019, a foliar disease with an incidence of ∼60% occurred on trees was observed in the soapberry germplasm repository, Jianning, Sanming, Fujian, China. The symptoms initially appeared as irregular small yellow spots, while the center of the lesions became dark brown with time. Fragments (size 3 to 4 mm2) taken from lesion margins were sterilized and cultured based on Wang et al. Two isolates (FJ1 and FJ21) were obtained with the following morphological characteristics on PDA, (1) FJ1: Conidiogenous cells were 9.7 to 25.0 × 1.5 to 2.2 μm (n=20). Alpha conidia were 6.1 to 8.3 × 2.2 to 3.0 μm (n=30), aseptate, hyaline, smooth, ellipsoidal. Beta conidia were 28.3 to 38.2 × 1.3 to 1.7 μm (n=30), hyaline, smooth, curved to hooked. Conidial drops were milky colored; (2) FJ21: Pycnidia were dark brown, 280 to 843 μm (n=30) in diam., globose, or irregular on alfalfa stems. Conidiophores were hyaline, cylindrical, smooth, and slightly tapered to the apex, 17.4 to 35.4 × 1.5 to 2.6 μm (n=20). Conidiogenous cells were 14.7 to 29.7 × 1.4 to 2.6 μm (n=20). Alpha conidia were 5.6 to 7.1 × 2.4 to 3.4 μm (n= 30), hyaline, smooth, ellipsoidal, or clavate, aseptate, biguttulate. Beta conidia not observed. Conidial drops were yellow. The morphological characteristics of FJ1 and FJ21 were similar to those of Diaporthe spp.. DNA of two isolates was extracted, and the internal transcribed spacer region (ITS) and partial sequences of translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), β-tubulin (TUB), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R, CAL228F/CAL737R, βt2a/βt2b, and CYLH3F/H3-1b, respectively. The sequences were deposited in GenBank (accession nos. MW585608 and MW768905 to MW768908 for FJ1; MT755625 and MT776728 to MT776731 for FJ21). The BLASTn results showed that the ITS, TEF1-α, TUB, HIS, and CAL sequences of FJ1 were 100, 99, 98, 98, and 99% identical to those of D. eres (NR144923, KJ210550, KJ420799, KJ420850, and KJ434999, respectively). For FJ21, BLASTing with the same loci showed 100, 100, 100, 99, and 100% similarity with those of D. unshiuensis (MH121530, MH121572, MH121607 MH121488, and MH121448, respectively). Phylogenetic analyses with the concatenated sequences placed FJ1 and FJ21 in the clades of D. eres and D. unshiuensis, respectively. Pathogenicity tests were performed by wounding leaves of 2-year-old soapberry seedlings with a sterile needle. The leaves were inoculated with D. eres and D. unshiuensis isolates, respectively, with 10 µl of conidial suspensions (106 conidia/ml). Three plants were used for each treatment, and the leaves of each plant were inoculated. The control was treated with 10 µl of sterile water. The plants were kept in a greenhouse (RH > 80%, 25 ± 2°C). In 5 days, all inoculated leaves showed lesions similar to the field symptoms. Controls were asymptomatic. Diaporthe eres and D. unshiuensis were reisolated from the diseased leaves. No fungus was isolated from the control. Previously, D. biconispora and D. sapindicola were reported as the causal agents of soapberry, but this is the first report of D. eres and D. unshiuensis causing leaf spots on S. mukorossi in China. These data will help develop effective strategies for managing this disease.

Species description and phylogenetics of Oerstedia fuscosparsa sp. nov. (Nemertea: Monostilifera: Oerstediidae) from Japan

We provide a morphological and molecular-based description of the eumonostiliferous hoplonemertean Oerstedia fuscosparsa sp. nov. based on two specimens dredged from a depth between 11 and 18 m off Kouyatsu, Tateyama, Chiba, Japan. This Oerstedia differs from its congeners by having a deep brownish pigmentation in the head, four orange ocelli, and imprecise dorsal and ventral transverse bands composed of numerous small brown spots. One of the specimens, herein designated as the holotype, has deep brown spots situated mid-dorsally onto the transverse bands running throughout the body. These dorsal markings are absent in the other specimen designated as a paratype. Internally, the species has accessory lateral nerves, and the proboscis nerves are not distinct. Molecular phylogenetic analyses based on partial sequences of the 16S, 18S, 28S rRNA, cytochrome c oxidase subunit I, and histone H3 gene markers along with the sequences from 22 Oerstedia species available in public databases confirmed that our species is sister to Oerstedia phoresiae (Kulikova, 1987) and belongs to the recently established Paroerstediella clade within the genus Oerstedia.

Distinct roles for canonical and variant histone H3 lysine-36 in Polycomb silencing.

Polycomb complexes regulate cell type-specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. We show that mutants of Drosophila replication-dependent (H3.2K36R) or replication-independent (H3.3K36R) histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined (H3.3K36RH3.2K36R) mutants display widespread Hox gene misexpression and fail to develop past the first larval stage. Chromatin profiling revealed that the H3.2K36R mutation disrupts H3K27me3 levels broadly throughout silenced domains, whereas these regions are mostly unaffected in H3.3K36R animals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive Polycomb response elements located outside of silenced domains but relatively depleted from those inside. We conclude that H3.2 and H3.3 K36 residues collaborate to repress Hox genes using different mechanisms.

First Report of Cercospora apii sensu lato 'sp. Q' Leaf Spot on Hop in Brazil.

Hop - Humulus lupulus (Cannabaceae) - is an important and ancient, herbaceous, temperate, perennial crop. It is a vine which, although having other uses, is cultivated predominantly for the brewing industry (Laws 2013). Cultivation of hops in Brazil is relatively new and has increased in the last twenty years. There is only one published record of a fungal disease affecting hop in Brazil - powdery mildew (Fagherazzi et al 2021). In January 2021, leaf spots appeared on all eight hops plants ithe collection maintained in the Infectarium, a disease demonstration garden on the campus of the Universidade Federal de Viçosa (UFV), Viçosa, Minas Gerais, Brazil (https://www.infectario.ufv.br/). Symptoms were small, sub-circular to irregular spots, up to 5 mm, with a whitish to grayish center, surrounded by a dark brown necrotic margin, followed by a narrow yellowish outer margin. Older lesions became larger, grayish-brown, coalesced leading to extensive necrosis and stem dieback. A sample was collected, dried in a plant press, and deposited in the Herbarium of UFV (Acc. No VIC 47534). A dematiaceous fungus was found sporulating in the center of the lesions, when examined with a dissecting microscope. Fungal structures were scraped with a scalpel, mounted in lactoglycerol and observed with a light microscope (Olympus BX51). A pure culture was obtained after conidia were transferred onto PDA plates with a sterile fine-pointed needle. A representative isolate was deposited in the culture collection of the UFV (COAD 3368). The fungus had the following morphology: conidiophores cylindrical, geniculate, proliferating sympodially, 53 to 380 µm × 3 to 6.5 µm, 3 to 15-septate, smooth, with thickened and dark conidial scars, brown; conidia acicular to filiform, 47 to 210 µm × 2.5 to 5 µm, 3 to 17-septate, thin‑walled, smooth, with thickened and dark hila, hyaline. This combination is typical of Cercospora apii sensu lato, as described by Crous and Braun (2003). Genomic DNA was extracted from a 7-day-old culture of COAD 3368 and three loci were PCR amplified, namely: actin (ACT), with 512-F and 783-R primers; calmodulin (CAL), using the primers 228F and 2Rd; and histone H3 gene (HIS3), with the primers CYLH3F and CYLH3R. PCR products were sequenced by Macrogen (Seoul, South Korea) and the resulting sequences were deposited in GenBank (OM677624, OM677625 and OM677626, respectively). A multilocus Bayesian phylogenetic grouped COAD 3368 with Cercospora 'sp. Q' (Groenewald et al. 2013). This is a yet unresolved species complex within C. apii. Six 6‑month‑old healthy hop plants (cv. Cascade) were sprayed with a COAD 3368 conidial suspension (105 conidia/ mL) whereas another group of six plants was sprayed with sterile distilled water, serving as controls. Plants were placed in a dew chamber for 5 days and then transferred to a greenhouse bench, where they were observed daily. Thirty days after inoculation, symptoms similar to those observed in the field had developed on all inoculated plants, whereas control plants remained healthy. The fungus growing on inoculated hops produced typical Cercospora conidiophores and conidia. Upon reisolation, pure cultures with the same morphology of COAD 3368 were obtained. There are no previous records of Cercospora 'sp. Q' on hops worldwide. Fungi in Cercospora 'sp. Q' are known to have a broad host-range. A previous record of Cercospora 'sp. Q' leaf spots was published from its observation on Dioscorea cayennensis in the Infectarium (Torres et al. 2016). Damage to the hop plants was severe and it is possible that Cercospora leaf spot will become an emerging threat to commercial hop plantations in Brazil.

First Report of Postharvest Fruit Brown Rot Disease on Navel Orange Caused by Diaporthe sojae in China.

In October 2020, a postharvest fruit brown rot symptom was observed on navel orange (Citrus sinensis Osbeck cv. Newhall) fruits in a local fruit market in Ganzhou, Jiangxi Province, China. The disease incidence increased up to 15% in 40 fruits with a 7-day-long storage at room temperature. The disease symptoms on the infected fruit were circular, light brown to brown, slightly sunken lesions, covered with whitish mycelium mass, and brown rot in the center. To isolate the causal organism, infected fruits were surface sterilized with 1% NaClO solution for 30 sec, and rinsed thrice with sterilized water. Symptomatic tissues at the margins were cut into 5-mm2 pieces, placed on potato dextrose agar (PDA) medium and incubated at 25℃ for 5 days. Thirteen morphologically similar single-spore fungal isolates were obtained from the isolation experiment. Fungal colonies were white, fluffy, cottony texture, reverse buff to light yellow, with black stromata at maturity. Alpha conidia were hyaline, aseptate, ellipsoid to clavate, tapering towards the ends, often biguttulate, and ranged in size from 6.8 to 9.8 µm × 2.7 to 4.5 µm (n=50). Beta conidia were hyaline, aseptate, smooth, straight to sinuous, and with size ranging from 12.1 to 21.3 µm × 0.9 to 2.2 µm (n=50). Morphological features were consistent with those of Diaporthe sojae (Dissanayake et al. 2015). For molecular identification, DNA was extracted from the representative isolate JFRL 03-13, the internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α), and histone H3 (HIS) genes were amplified by using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, and CYLH3F/H3-1b (Udayanga et al. 2015), respectively. The resulting sequences were deposited in GenBank (Accession Nos. OM281710 for ITS, OM289961 for TUB, OM289964 for CAL, OM289963 for TEF1-α, and OM289962 for HIS). BLAST analysis revealed that these sequences were 100% similar to the sequences of ITS (MN816426), TUB (MK941336), CAL (MN894375), TEF1-α (MN894447), HIS (MN894409) published for D. sojae. Phylogenetic analysis was conducted based on the concatenated sequences (ITS, TUB, CAL, TEF1-α, and HIS) by Maximum likelihood analysis (ML) and Bayesian inference (BI) using IQtree v.1.6.11 and MrBayes v.3.2.7 (Guo et al. 2020). The phylogenetic tree showed that the isolate clustered with D. sojae. To confirm pathogenicity, mature and healthy harvested fruits of navel orange (Citrus sinensis Osbeck cv. Newhall) were surface sterilized. Ten fruits were wounded by a sterile scalpel and put a 7-mm-diamter agar plug with 7-day-old mycelium of the isolate JFRL03-13 cultured on PDA at 25°C, noncolonized PDA plugs were used as the control. Inoculated fruits were incubated at 25℃ with 80% relative humidity. After 10 days, the similar symptoms were observed on the inoculated sites and spread on the surface of fruits, whereas the control remained symptomless. The pathogen was re-isolated from the lesions of inoculated fruits and confirmed as D. sojae via morphological and molecular analysis. The assays were repeated twice, fulfilling the Koch's postulates. Although D. sojae is known as the major causative agent of pod and stem blight, and has been reported as an endophyte in the twigs and leaves of citrus (Huang et al. 2015; Santos et al. 2011), but to our knowledge, this is the first report of postharvest fruits brown rot disease on navel orange caused by D. sojae in China. However, further investigation of the specific causes of this disease is necessary to help the local fruit farmers develop effective disease management strategies.

Calonectria queenslandica: Causal Agent of Eucalyptus Leaf Blight in Southern China.

Calonectria leaf blight caused by Calonectria spp. is among the most serious diseases affecting the health and sustainability of Eucalyptus plantations in southern China. Recent outbreaks of this disease in GuangDong Province prompted a need to identify the species involved. Typical symptoms of Calonectria leaf blight were observed on 2-year-old Eucalyptus urophylla × E. grandis trees in a plantation in the ZhaoQing region. In total, 38 Calonectria isolates were collected from 32 diseased trees. All isolates were identified using DNA sequence analyses of the translation elongation factor 1-α (tef1), β-tubulin (tub2), calmodulin (cmdA), and histone H3 (his3) gene regions. Phylogenetic analyses revealed that Calonectria queenslandica was the dominant species, accounting for 81.6% of the isolates collected. Other species isolated included C. pseudoreteaudii (10.5%), C. reteaudii (5.3%), and C. aconidialis (2.6%). This is the first report of C. queenslandica in China and all isolates had identical sequences in all four gene regions. PCR amplification using primers targeting the MAT1-1-1 and MAT1-2-1 genes in all C. queenslandica isolates revealed that only the MAT1-2 idiomorph was present. The results suggest that C. queenslandica was introduced into the sampled area with very limited genetic diversity. Pathogenicity tests were conducted on two Eucalyptus genotypes widely planted in the GuangDong Province using isolates representing all species collected. The results showed that these species could all cause disease but the predominance of C. queenslandica on infected trees suggests that it is the major driver of the disease problem studied. Different Eucalyptus genotypes used in the pathogenicity tests differed in susceptibility to infection by the Calonectria spp. tested, providing opportunities to avoid leaf blight by deploying disease-tolerant planting stock.

Phylogenetic and morphological analyses of Japanese Aquarius water strider, with a new synonym

The genus Aquarius, of the subfamily Gerrinae, was formerly divided into two species and one subspecies in Japan (A. paludum paludum, A. paludum amamiensis and A. elongatus, respectively). Aquarius haliplous Yasunaga et al. 2018 has recently been described as a new species, which occurs in brackish waters of Nagasaki Prefecture and is found sympatrically with the widely distributed species, A. p. paludum. However, they are considered to be speciated, with no cross-breeding. In this study, molecular phylogenetic and morphological analyses were undertaken using the mitochondrial DNA COI (529 bp) and nuclear DNA histone H3 (314 bp) gene regions to investigate the genetic and morphological differentiation in the genus Aquarius, including A. haliplous, throughout Japan (including the islands). The results show that, unlike A. p. paludum, A. p. amamiensis and A. haliplous, A. elongatus displays substantial genetic differentiation. Considering that, although morphological differences exist in the antennae of A. p. paludum and A. p. amamiensis, A. haliplous has no clear genetic or morphological difference from A. p. paludum, and hence cannot be classified as a distinct species. Therefore, we propose that A. haliplous be synonymized with A. paludum.

First Report of Diaporthe eres Causing Leaf Spot of Viburnum odoratissimum var. awabuki in China.

Viburnum odoratissimum var. awabuki (K. Koch) Zabel ex Rumpl. is an evergreen tree, used as a landscape plant in China. In June 2019, a foliar disease of ~60% incidence was observed on V. odoratissimum var. awabuki at the campus of Nanjing Forestry University, Jiangsu, China. The symptoms were initially irregular small red-brown spots, later enlarged and became brown to black. Small pieces of tissue (3 to 4 mm2) cut from lesion margins were surfaced sterilized in 75% ethanol for 30 s and 1.5% NaClO for 60 s, then rinsed in sterile water and placed on potato dextrose agar (PDA) at 25℃. Pure cultures were obtained from the tip of hyphae. Using the standard phytopathological procedure, two representative isolates (SH161 and SH181) were obtained and deposited at Nanjing Forestry University. The colony on PDA was white with aerial mycelium, radiate, and the reverse was white. Black pycnidia developed on the sterilized alfalfa stems at 25°C with a 14/10 h light/dark cycle for 20 days. Conidiophores were hyaline, branched, straight to sinuous, 9.4 to 26.0 × 1.0 to 2.5 μm (n=30). Conidiogenous cells were 2.1 to 15.1 × 0.9 to 2.5 μm (n=30). Alpha conidia were 7.4 ± 0.6 × 2.0 ± 0.2 μm (n=50), hyaline, ellipsoidal to lanceolate. Beta conidia were 29.5 ± 1.8 × 1.1 ± 0.1 μm (n=30), aseptate, hyaline, smooth, curved to hooked. Morphological features of two isolates matched those of Diaporthe spp.. DNA of two isolates was extracted and the internal transcribed spacer region (ITS), partial translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), beta-tubulin (TUB), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R, CAL228F/CAL737R, βt2a/βt2b and CYLH3F/H3-1b. The sequences were deposited into GenBank (Accession Nos. for isolate SH161: OK326730 for ITS, OK413403 to OK413406 for TUB, CAL, HIS and TEF1-α; and isolate SH181: OK331347 for ITS, OK413407 to OK413410 for TUB, CAL, HIS, and TEF1-α). BLAST search of SH161 showed high similarities with sequences of Diaporthe eres (AR5193) [KJ210529 (ITS), Identities = 438/512, (94%); KJ420850 (HIS), Identities = 466/472, (99%); KJ210550 (TEF1-α), Identities = 345/350, (99%); KJ434999 (CAL), Identities = 344/345, (99%); KJ420799 (TUB), Identities = 508/517, (98%)]. BLAST results of SH181 are listed in Supplementary Table 1. Maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and MrBayes v. 3.2.6 with the concatenated sequences placed SH161 and SH181 in the clade of D. eres. Based on the multi-locus phylogeny and morphology, two isolates were identified as D. eres. The pathogenicity was tested on 1-yr-old cuttings of V. odoratissimum var. awabuki in the greenhouse. Healthy leaves were wounded with a sterile needle, then inoculated with 5-mm plugs from the edge of two isolates cultures. The PDA plugs were used for controls. Three plants were used for each treatment, and three leaves of each plant were inoculated. Each plant was covered with a plastic bag, and sterilized water was sprayed into the bags bidaily to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2°C/16 ± 2°C. Three days after inoculation, the inoculated leaves appeared lesions similar to those in the field. The controls remained healthy. Diaporthe eres was reisolated from inoculated leaves. No fungus was isolated from controls. Diaporthe eres was reported from Viburnum lantana in Austria. Also, it was reported from V. odoratissimum and V. tinus in Ukraine. This is the first report of D. eres causing V. odoratissimum var. awabuki leaf spots in China. This finding will provide an effective basis for developing control strategies for the disease.

The Intertwined Effects of Hydrographic Barriers, Palaeoclimate and Life History on Genetic Structure of Marine Populations: A Case Study of Two Marine Invertebrates

Marine organisms with pelagic planktonic larval stages have high dispersal potential, yet their ranges are restricted by present and historical geographic and oceanographic features. Hydrogeographic features, such as straits and water current patterns, as well as paleoclimate can affect population connectivity, genetic differentiation, and ultimately speciation, but species distributions are also affected by life history characteristics. This study evaluates the effect of the Turkish Straits System (TSS) on the genetic differentiation of two benthic invertebrates with pelagic larvae, the Mediterranean mussel, Mytilus galloprovincialis, and the rockpool prawn, Palaemon elegans, using mitochondrial and nuclear markers (microsatellites for M. galloprovincialis and Histone H3 gene for P. elegans). For both species, the mitochondrial DNA analyses separated the Black Sea and the Eastern Mediterranean (Aegean and Levantine seas) populations into two clusters. In contrast, in both species the nuclear data indicated no differentiation between Black Sea and Eastern Mediterranean populations. The results suggest that for both species, some individuals of Black Sea origin moved south, most likely through the transport of their pelagic larvae via the surface currents of the TSS. However, for the most part, individuals of Mediterranean origin (from Aegean and Levantine seas) were not able to successfully migrate in the opposite direction, though the small differences in the frequencies of migration in this direction indicate the effects of the life history characteristics of the two species on genetic structure.