Post Inoculation Research Articles

Comparative transcriptomic and physiological analyses reveal the key role of abscisic acid in hydrangea macrophylla responding to Corynespora cassiicola.

Bigleaf hydrangea (Hydrangea macrophylla) is a widely cultivated ornamental plant species. Leaf spot disease, caused by Corynespora cassiicola, poses a significant threat to the ornamental quality and economic value of hydrangeas. However, the disease resistance breeding of hydrangea is limited due to the lacking of resistant varieties and genes. This study evaluated ten hydrangea varieties for their resistance to leaf spot disease. Among them, 'White Angel' and 'Ocean Heart' were screened out as representative varieties for resistance and susceptibility, respectively, on the basis of evaluation. Physiological and biochemical indices, phytohormones, and transcriptomic changes were measured in the leaves of both varieties at 0 and 24h post inoculation with C. cassiicola. The results showed that C. cassiicola infection significantly increased abscisic acid (ABA) contents in both varieties; however, the increase was significantly higher in the susceptible variety 'Ocean Heart' compared to the resistant variety 'White Angel' (p < 0.05). Moreover, exogenous ABA (100 µM) decreased the leaves' resistance to C. cassiicola of both varieties, underscoring its key role in reduced disease resistance. Transcriptome profiling revealed 17,087 differentially expressed genes (DEGs) responding to C. cassiicola between the two varieties. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated significant enrichment of DEGs in "Plant hormone signal transduction", particularly related to ABA signaling (HmPP2C and HmABFs). In addition, the expression of ABA biosynthesis genes (HmZEP3, HmABA2, and HmAAO3) was upregulated in both varieties. Meanwhile, the ABA catabolism gene (HmCYP707A4) exhibited significantly upregulated expression in the resistant variety 'White Angel' and downregulated expression in the susceptible variety 'Ocean Heart'. Intriguingly, the expression of HmCYP707A4 was 15-fold higher in 'White Angel' than in 'Ocean Heart'. In summary, these findings highlight the crucial role of ABA in the resistance of bigleaf hydrangea to leaf spot disease and provide valuable genetic resources for breeding programs to enhance the disease resistance in hydrangeas.

Exploring common bean's defense arsenal: Genome-wide characterization of PR-1 gene family and its transcriptional response to Colletotrichum lindemuthianum inoculation

Pathogenesis-related Protein 1 (PR-1) plays a crucial role in plant defense responses, particularly against fungal pathogens. Despite its significance, comprehensive studies characterizing this gene family in the common bean (Phaseolus vulgaris) are currently lacking. Therefore, the objective of this study was to conduct genomic mining and characterization of the PR-1 in common bean (PvPR-1) genome. Additionally, we assessed the transcriptional expression of all its isoforms in response to inoculation with the fungus Colletotrichum lindemuthianum. This evaluation was performed on leaf tissue samples obtained from both sensitive (Rosinha) and resistant (Africano 4) common bean varieties at 24-, 48-, and 96-hours post inoculation. Thirteen PvPR-1 genes were consistently identified, forming two major clusters across the clustering analyses. Physicochemical characterization indicated that the PvPR-1 proteins are predominantly basic, hydrophilic, and extracellularly localized. Moreover, their promoter regions contain putative cis-regulatory elements that respond to a broad spectrum of plant hormones, including jasmonic acid, gibberellin, and ethylene, which are key regulators of both biotic and abiotic stress responses. This discovery implies a multifaceted role for the studied proteins in common bean physiology. KEGG pathway analysis implicated PvPR-1 proteins in hormonal signaling (corroborating the anchored cis-regulatory elements) and plant-pathogen interaction networks. Secondary structure evaluation revealed the predominance of α-helices and coiled structures within these proteins. Subsequent 3D modeling demonstrated a conserved ‘α-β-α’ sandwich architecture characterized by a central cavity. This structural motif suggests potential functional versatility, particularly in pathogen recognition and responses. Additionally, the study provided insight into the potential interactions of PvPR-1 with Chitinase II (PR-3) and Rab-18, as suggested by the STRING platform. Temporal differences in PvPR-1 gene expression were observed between the common bean contrasting varieties following C. lindemuthianum inoculation. Africano-4, the resistant one, showed a higher abundance of up-regulated and constitutively expressed PvPR-1 transcripts compared to its sensitive counterpart (Rosinha), indicating a more effective role of this gene family against the pathogen. Furthermore, based on PCA analyses and interaction networks of differentially expressed genes, three key targets within the PvPR-1 family (PvPR-1-4, PvPR-1-5, and PvPR-1-10) emerged as promising candidates for future functional characterization. These molecular actors displayed differential transcriptional patterns between the studied varieties without compromising the transcript abundance of PvPR-1 protein synthesis in the resistant one. Consequently, they may represent key components of resistance mechanisms that contribute to the differentiation between the two organisms. These findings deepen our understanding of PvPR-1 genes and their roles in common bean defense responses. They also emphasized the potential of PvPR-1 genes as candidates for breeding stress-resistant common bean varieties, which are crucial for bolstering crop resilience to environmental adversities.

Dietary glycerides of valerate ameliorate diarrhea and impact intestinal physiology and serum biomarkers in weaned piglets infected with enterotoxigenic Escherichia coli F18.

In the commercial swine farm setting, the post-weaning period is a critical window during which piglets are highly susceptible to infection and enterotoxigenic E. coli (ETEC)-associated diarrhea. Short chain fatty acids and their glycerides are compounds which may influence intestinal health; however, valerate is one that has not been well-characterized for its role as a dietary supplement. Therefore, the major objective of this experiment was to investigate two forms of valerate glycerides on diarrhea, intestinal physiology, and systemic immunity of weaned pigs experimentally infected with ETEC F18. Dietary treatments included a control diet and three additional diets supplemented with 0.075% monovalerin, 0.1% monovalerin, or 0.1% trivalerin, respectively. Piglets were weaned (21-24 d of age), individually housed, and experimental diets were fed throught the 28-day trial period. After a seven-day period, all piglets were inoculated on three consecutive days with 1010 CFU ETEC F18/3mL. Growth performance was monitored throughout the trial and daily diarrhea scores were recorded. Rectal swabs were collected for bacterial culture to confirm the presence or absence of β-hemolytic coliforms throughout the trial. Serum samples were collected and analyzed for inflammatory biomarkers on d 0, 3, 6, and 21 post-inoculation (PI) and untargeted metabolomics on d 6 PI. Intestinal mucosa and tissue sections were harvested from pigs sacrificed on d 7 PI for gene expression and histology analysis. All data, except for frequency of diarrhea and metabolomics, were analyzed by ANOVA using the PROC MIXED of SAS. Dietary trivalerin reduced (P < 0.05) the frequency of severe diarrhea over the entire trial period and the frequency of β-hemolytic coliforms on d 7 PI compared with control. The intestinal villus height on d 7 PI in jejunum tissue was increased (P < 0.05) in pigs fed trivalerin. The mRNA expression of TNF-α was decreased (P < 0.05) in the trivalerin group, while that of ZO1 was increased (P < 0.05) compared with control. Throughout the trial, serum TNF-α was reduced in pigs fed trivalerin compared with control. Serum metabolites, adenosine, inosine, and shikimic acid were reduced (P < 0.05) on d 6 PI in all treatment groups compared with control. In conclusion, the present results indicate supplementing dietary valerate glycerides exhibited beneficial impacts on diarrhea, inflammation, and intestinal gene expression of piglets during the post-weaning period.

First report of Sclerotinia minor causing soft rot of Scabiosa atropurpurea in California.

In May of 2019, Scabiosa atropurpurea samples with brown discoloration, soft rot of the crown and lower stem, with presence of white mycelium and black sclerotia (Supp. Fig. 1A, B) were collected from a 0.10 ha open field diversified cut flower production in San Luis Obispo County, CA. Approximately 30 to 40% of the scabiosa crop planted in a quarter of the field, exhibited symptoms. Symptomatic crowns and lower stems from five plants were surface disinfested by rinsing in 0.1% Tween 20, soaking in 70% ethanol for 30 s, 1% sodium hypochlorite for 2 min and sterile water. Disinfested tissue was placed in 1/10 potato dextrose agar (PDA) and incubated at 20°C (12 h photoperiod). Resulting colonies (n = 5) formed abundant white mycelia, with black sclerotia formed on the outer edge of the plates after two weeks (Suppl. Fig. 1C). Sclerotia (n = 50) had an average size of 1.6 (± 0.19) mm in diameter. Morphological identification resulted in Sclerotinia sp. (Hao et al., 2003). The pathogen was further identified by DNA extraction of two hyphal tipped isolates, followed by amplification and sequencing of the rDNA internal transcribed spacer (ITS) region, ITS1/ITS4 (White et al. 1990), calmodulin (CaM), CAL-228F/CAL-737R (Carbone and Kohn, 1999), and DNA replication licensing factor Mcm7 Mcm7-709for/Mcm7-1348rev (Schmitt et al., 2009). NCBI BLAST searches with consensus sequences for each maker revealed 99 to 100% identity with S. minor ex-types for all loci (Supp. Table 1). A maximum parsimony multilocus phylogenetic analysis clustered Californian isolates with reference strains of S. minor (Supp. Fig. 1F). Sequences were deposited in GenBank (Supp. Table 1). Pathogenicity tests were conducted with isolate CS435, which was transferred onto PDA plates and incubated at 20°C for one week. Inoculum consisted of CS435 infested PDA plugs (1 cm3). In the greenhouse, the experiment was set as a complete randomized design and observed for six weeks. Fourteen-week-old scabiosa 'Merlot Red' grown in 3.78 L pots (n = 6), were inoculated by wounding plants at 0.5 cm above the crowns with a 1 mm probe. Inoculum was placed directly on top of the wound and was secured with parafilm. Negative control plants (n = 6) were wounded as above and inoculated with PDA plugs. In experiment 1(19.4 (± 3) °C, RH 46.9), 83% of plants exhibited yellowing of the lower leaves and wilting at one week post inoculation (wpi). Symptoms progressed over time until wilting, major leaf and stem necrosis, was observed in all inoculated plants (Supp. Fig 1E, D). Plant mortality incidence at five wpi was 83%. Pathogen signs including white mycelia and black sclerotia were also observed. In experiment 2 (20.0 (± 10) °C, RH 39.6), 66% of the total plants were symptomatic at five wpi: 33% exhibited yellowing of the lower leaves and wilting, and 33% of plants died. Disease did not develop in non-inoculated plants in either experiment. S. minor was successfully reisolated from surface disinfested tissue of at least 50% (Exp. 1) and 100% (Exp. 2) of inoculated plants, yielding identical sequences to those of the inoculated isolate. S. minor is a soil-borne pathogen that infects tomato, lettuce, brassica, and sunflower crops in California. This study stablishes S. minor as a new soil-borne pathogen of Scabiosa in California. Soil-borne pathogens are a recurrent issue in the cut flower industry, characterizing new pathogens in these crops can inform crop advisors and disease diagnostician to improve disease management in the ornamental industry.

Transcriptomic insights into mycorrhizal interactions with tomato root: a comparative study of short- and long-term post-inoculation responses.

Arbuscular mycorrhiza (AM) refers to a symbiotic association between plant roots and fungi that enhances the uptake of mineral nutrients from the soil and enables the plant to tolerate abiotic and biotic stresses. Although previously reported RNA-seq analyses have identified large numbers of AM-responsive genes in model plants, such as Solanum lycopersicum L., further studies are underway to comprehensively understand the complex interactions between plant roots and AM, especially in terms of the short- and long-term responses after inoculation. Herein, we used RNA-seq technology to obtain the transcriptomes of tomato roots inoculated with the fungus Rhizophagus irregularis at 7 and 30days post inoculation (dpi). Of the 1,019 differentially expressed genes (DEGs) in tomato roots, 635 genes showed differential expressions between mycorrhizal and non-mycorrhizal associations at the two time points. The number of upregulated DEGs far exceeded the number of downregulated ones at 7 dpi, and this difference decreased at 30dpi. Several notable genes were particularly involved in the plant defense, plant growth and development, ion transport, and biological processes, namely, GABAT, AGP, POD, NQO1, MT4, MTA, and AROGP3. In addition, the Kyoto encyclopedia of genes and genomes pathway enrichment analysis revealed that some of the genes were involved in different pathways, including those of ascorbic acid (AFRR, GME1, and APX), metabolism (CYP, GAPC2, and CAM2), and sterols (CYC1 and HMGR), as well as genes related to cell division and cell cycle (CDKB2 and PCNA). These findings provide valuable new data on AM-responsive genes in tomato roots at both short- and long-term postinoculation stages, enabling the deciphering of biological interactions between tomato roots and symbiotic fungi.

Dual transcriptional characterization of spinach and Peronospora effusa during resistant and susceptible race-cultivar interactions

BackgroundSpinach downy mildew, caused by the obligate oomycete pathogen, Peronospora effusa remains a major concern for spinach production. Disease control is predominantly based on development of resistant spinach cultivars. However, new races and novel isolates of the pathogen continue to emerge and overcome cultivar resistance. Currently there are 20 known races of P. effusa. Here we characterized the transcriptomes of spinach, Spinacia oleracea, and P. effusa during disease progression using the spinach cultivar Viroflay, the near isogenic lines NIL1 and NIL3, and P. effusa races, R13 and R19, at 24 h post inoculation and 6 days post inoculation. A total of 54 samples were collected and subjected to sequencing and transcriptomic analysis.ResultsDifferentially expressed gene (DEG) analysis in resistant spinach interactions of R13-NIL1 and R19-NIL3 revealed spinach DEGs from protein kinase-like and P-loop containing families, which have roles in plant defense. The homologous plant defense genes included but were not limited to, receptor-like protein kinases (Spiol0281C06495, Spiol06Chr21559 and Spiol06Chr24027), a BAK1 homolog (Spiol0223C05961), genes with leucine rich repeat motifs (Spiol04Chr08771, Spiol04Chr01972, Spiol05Chr26812, Spiol04Chr11049, Spiol0084S08137, Spiol03Chr20299) and ABC-transporters (Spiol02Chr28975, Spiol06Chr22112, Spiol06Chr03998 and Spiol04Chr09723). Additionally, analysis of the expression of eight homologous to previously reported downy mildew resistance genes revealed that some are differentially expressed during resistant reactions but not during susceptible reactions. Examination of P. effusa gene expression during infection of susceptible cultivars identified expressed genes present in R19 or R13 including predicted RxLR and Crinkler effector genes that may be responsible for race-specific virulence on NIL1 or NIL3 spinach hosts, respectively.ConclusionsThese findings deliver foundational insight to gene expression in both spinach and P. effusa during susceptible and resistant interactions and provide a library of candidate genes for further exploration and functional analysis. Such resources will be beneficial to spinach breeding efforts for disease resistance in addition to better understanding the virulence mechanisms of this obligate pathogen.

The Direct Gene Injection Using of Egg Yolk Emulsion as Carrier and its Comparison with Liposomes.

The efficiency of egg yolk emulsion in coating DNA and its delivery across cellular membranes was evaluated in comparison with liposomes DOPE . The murine leukemia viral oncogene v-abl , cloned on pBR322 was used as a DNA substrate for direct injection into mice tissue . the DNA complexes were prepared by mixing the DNA with egg yolk emulsion and liposome . Each was directly injected into mice peritoneal cavity with proper control. The gene delivery was examined phenotypically by blood analysis and cytogenetic analysis . Chromosomal changes were detected in the bone marrow as from the fourth day post inoculation through the eleventh day when chromosomal ring s could be seen . this was accompanied by decrease in the WBC count , an increase in lymphoblast cells size and percentage and the discoloration of irregular RBCs . these are typical signs of acute lymphatic leukemia . Anatomical examination have indicated 25% increase in the spleen mplemented using specific primers to confirm the integration of the delivered DNA into the genomic content of the mice tissue within 48 hr post inoculation .

Exposure dose, light distribution and wavelength affect the fate of introduced bacterial biological control agents in the phyllosphere of greenhouse grown tomato

Societal Impact StatementThe use of chemical plant protection products must be reduced to promote sustainability in food production. One possible alternative is biological control agents (BCAs), but their efficacy under commercial conditions does not always reach the standard of chemical control agents. Previously, light has been found to induce mechanisms in bacterial BCAs that can affect their distribution and establishment. This could promote BCA efficacy. We looked into how monochromatic and polychromatic (which is what growers use) light treatments affected the occurrence of three BCAs post‐application. By combining two non‐chemical methods: a biological (BCA) and a cultural (light) control method, this offers a new integrated pest control strategy.Summary The dynamics and functionality of beneficial and non‐beneficial, non‐phototrophic bacteria can be influenced by light quality. We investigated if light could aid the survival of three bacterial biological control agents (BCAs; Bacillus amyloliquefaciens DSM7, Pseudomonas chlororaphis 50083 and Streptomyces griseoviridis CBS904.68) in the canopy of greenhouse‐grown tomatoes at four light treatments. Tomato plants were exposed to 50 μmol m−2 s−1 of either polychromatic light (white) or monochromatic light (blue: 420 nm, green: 530 nm and red: 660 nm) using DYNA LED lamps for a total of 48 h post foliar application of the BCAs. Leaves were harvested from two levels in the canopy at the top and middle of each plant at 0, 2, 4, 8, 12, 24 and 48 h post inoculation. The occurrences of the BCAs were quantified by plate count and droplet digital PCR (ddPCR). S. griseoviridis persisted under most treatments, whereas P. chlororaphis and B. amyloliquefaciens preferred the polychromatic and green light treatments as depicted by the viable count analyses. Significant differences between the DNA and cDNA concentrations were only noted for P. chlororaphis, with prominent wavelength effects. Light exposure dose, placement in the canopy and wavelength were found to be decisive factors for BCA re‐isolation, indicating that they have different optimal light environments.

Biochemical Defense Arsenal, Genes/QTLs and Transcripts for Imparting Anthracnose Resistance in Common bean (Phaseolus vulgaris L.)

Anthracnose (ANT), caused by Colletotrichum lindemuthianum, is the most devastating disease affecting common bean (Phaseolus vulgaris L.), leading to significant yield losses in the Western Himalayas. The study provides a comprehensive understanding of ANT resistance via trait phenotyping, biochemical profiling, genome-wide association studies (GWASs), and RNA sequencing. The assessment of bean association mapping panel in different environments revealed a diverse spectrum of resistance levels. Biochemical analysis revealed distinctive defense responses against ANT infection among different genotypes. GWAS approach identified 24 significant marker‒trait associations (MTAs) distributed across all 11 bean chromosomes. Notably, 03 MTAs (BMr205, BMr269 and BMr244) present on chromosome Pv07 were validated for ANT, and the remaining MTAs were novel MTAs for ANT. Transcriptome sequencing of resistant (PBG-3) and susceptible (PBG-26) genotypes under mock and 120-hour post inoculation conditions revealed key differentially expressed genes, such as leucine-rich repeat domain-containing protein (PHAVU_007G087700g), NB-ARC domain-containing protein (PHAVU_003G002500g) and transcription factors pivotal for disease resistance. The expression patterns of four genes (PHAVU_007G087700g, PHAVU_003G002500g, PHAVU_007G056100g and PHAVU_003G003000g) were validated through quantitative reverse transcription polymerase chain reaction (qRT‒PCR). Furthermore, the integration of GWAS-identified candidate genes with transcriptomics and cross-referencing with previous studies validated overlapping regions and common candidate genes, enriching our understanding of the genetic basis of ANT resistance. Therefore, the results offer a holistic perspective on ANT resistance in common bean, providing a foundation for targeted breeding efforts. The identified potential candidate genes and associated pathways will contribute valuable insights into the development of ANT resistant common bean varieties.

A Comparison of the Cecal Microbiota between the Infection and Recovery Periods in Chickens with Different Susceptibilities to Eimeria tenella.

To investigate the effect of Eimeria tenella (E. tenella) infection on the cecal microbiota, resistant and susceptible families were screened out based on the coccidiosis resistance evaluation indexes after E. tenella infection. Subsequently, a comparative analysis of cecal microorganisms among control, resistant, and susceptible groups as well as between different periods following the E. tenella challenge was conducted using metagenomic sequencing technology. The results showed that the abundance of opportunistic pathogens, such as Pantoea, Sporomusa, and Pasteurella in the susceptible group and Helicobacter and Sutterella in the resistant group, was significantly higher on day 27 post-inoculation (PI) (the recovery period) than on day 5 PI (the infection period). Additionally, the abundance of Alistipes, Butyricicoccus, and Eubacterium in the susceptible group and Coprococcus, Roseburia, Butyricicoccus, and Lactobacillus in the resistant group showed a significant upward trend during the infection period compared with that in the recovery period. On day 5 PI, the abundance of Faecalibacterium and Lactobacillus was decreased in both the resistant and susceptible groups when compared with that in the control group and was greater in the resistant group than in the susceptible group, while Alistipes in the susceptible group had a relatively higher abundance than that in other groups. A total of 49 biomarker taxa were identified using the linear discriminant analysis (LDA) effect size (LEfSe) method. Of these, the relative abundance of Lactobacillus aviarius, Lactobacillus salivarius, Roseburia, and Ruminococcus gauvreauii was increased in the resistant group, while Bacteroides_sp__AGMB03916, Fusobacterium_mortiferum, Alistipes_sp__An31A, and Alistipes_sp__Marseille_P5061 were enriched in the susceptible group. On day 27 PI, LDA scores identified 43 biomarkers, among which the relative abundance of Elusimicrobium_sp__An273 and Desulfovibrio_sp__An276 was increased in the resistant group, while that of Bacteroides_sp__43_108, Chlamydiia, Chlamydiales, and Sutterella_sp__AM11 39 was augmented in the susceptible group. Our results indicated that E. tenella infection affects the structure of the cecal microbiota during both the challenge and recovery periods. These findings will enhance the understanding of the effects of changes in the cecal microbiota on chickens after coccidia infection and provide a reference for further research on the mechanisms underlying how the intestinal microbiota influence the growth and health of chickens.

Immune modulation by dexketoprofen trometamol, a selective eicosanoid biosynthesis inhibitor of cellular immune response and phenoloxidase reaction in response to viral infection in Pimpla turionellae adults

Nodulation is the first immune defence mechanism related to melanisation in response to microbial infections in insects. Adult parasitoid insects have been hypothesised to produce nodules with melanisation in response to viral infections and, eicosanoids, to mediate nodulation reactions and phenoloxidase (PO) activation in this type of infections. To test this hypothesis, endoparasitoid Pimpla turionellae adults were first inoculated with a novel generation nonsteroidal anti-inflammatory drug (NSAID) dexketoprofen trometamol (DT) (5 μg/adult), which is a selective cyclooxygenase-1 (COX-1) inhibitor. These adults were then immediately injected with intrahaemocoelic injection of Bovine herpes simplex virus-1 (BHSV-1) as a model insect-virus interaction. Additionally, adults were fed on artificial diet with increasing concentrations of DT (0.001, 0.01, or 0.1 g/100 ml diet) per os prior to intrahaemocoelic injection of BHSV-1 (2 × 103 PFU/adult) and nodulation and PO activity were recorded at 2 h post inoculation (PI). BHSV-1-treated newly emerged adults fed with inhibitors showed low levels of nodulation and increased PO enzyme activity. DT-treated Pimpla adults produced significantly fewer nodules (approximately nine nodules/adult), whereas viral infection provoked nodules (approximately 33 nodules/adult) in comparison with needle (vehicle)-treated controls (approximately five nodules/adult). Increasing dietary dexketoprofen trometamol concentrations decreased nodulation (by 12-fold at the highest concentration) and increased PO reactions (by approximately 3-fold at the highest concentration) to BHSV-1 injection. Compared with control adults, adults orally fed on the lowest DT concentration (0.001 %) significantly increased PO activity (1.22 ± 0.23–2.74 ± 0.31 unit/min/mg protein) while nodules significantly decreased (43.19 ± 4.26–17.84 ± 2.19) in response to virus infections. These findings suggest that eicosanoid biosynthesis, at least in the context of prostaglandins (PGs) formed by COX-1, mediates nodulation reactions and PO activation in response to viral infection in adults of this endoparasitoid. This is the first demonstration that the immune response of P. turionellae adults to viral pathogens is modulated by DT, which initiates haemolymph PO activation.

Effector Pt9226 from Puccinia triticina Presents a Virulence Role in Wheat Line TcLr15.

Effectors are considered to be virulence factors secreted by pathogens, which play an important role during host-pathogen interactions. In this study, the candidate effector Pt9226 was cloned from genomic DNA of Puccinia triticina (Pt) pathotype THTT, and there were six exons and five introns in the 877 bp sequence, with the corresponding open reading frame of 447 bp in length, encoding a protein of 148 amino acids. There was only one polymorphic locus of I142V among the six Pt pathotypes analyzed. Bioinformatics analysis showed that Pt9226 had 96.46% homology with the hypothetical putative protein PTTG_26361 (OAV96349.1) in the Pt pathotype BBBD. RT-qPCR analyses showed that the expression of Pt9226 was induced after Pt inoculation, with a peak at 36 hpi, which was 20 times higher than the initial expression at 0 hpi, and another high expression was observed at 96 hpi. No secretory function was detected for the Pt9226-predicted signal peptide. The subcellular localization of Pt9226Δsp-GFP was found to be multiple, localized in the tobacco leaves. Pt9226 could inhibit programmed cell death (PCD) induced by BAX/INF1 in tobacco as well as DC3000-induced PCD in wheat. The transient expression of Pt9226 in 26 wheat near-isogenic lines (NILs) by a bacterial type III secretion system of Pseudomonas fluorescens EtHAn suppressed callose accumulation triggered by Ethan in wheat near-isogenic lines TcLr15, TcLr25, and TcLr30, and it also suppressed the ROS accumulation in TcLr15. RT-qPCR analysis showed that the expression of genes coded for pathogenesis-related protein TaPR1, TaPR2, and thaumatin-like protein TaTLP1, were suppressed, while the expression of PtEF-1α was induced, with 1.6 times at 72 h post inoculation, and TaSOD was induced only at 24 and 48 h compared with the control, when the Pt pathotype THTT was inoculated on a transient expression of Pt9226 in wheat TcLr15. Combining all above, Pt9226 acts as a virulence effector in the interaction between the Pt pathotype THTT and wheat.

Transcriptome analysis of resistant and susceptible Medicago truncatula genotypes in response to spring black stem and leaf spot disease

Ascochyta blights cause yield losses in all major legume crops. Spring black stem (SBS) and leaf spot disease is a major foliar disease of Medicago truncatula and Medicago sativa (alfalfa) caused by the necrotrophic fungus Ascochyta medicaginicola. This present study sought to identify candidate genes for SBS disease resistance for future functional validation. We employed RNA-seq to profile the transcriptomes of a resistant (HM078) and susceptible (A17) genotype of M. truncatula at 24, 48, and 72 h post inoculation. Preliminary microscopic examination showed reduced pathogen growth on the resistant genotype. In total, 192 and 2,908 differentially expressed genes (DEGs) were observed in the resistant and susceptible genotype, respectively. Functional enrichment analysis revealed the susceptible genotype engaged in processes in the cell periphery and plasma membrane, as well as flavonoid biosynthesis whereas the resistant genotype utilized calcium ion binding, cell wall modifications, and external encapsulating structures. Candidate genes for disease resistance were selected based on the following criteria; among the top ten upregulated or downregulated genes in the resistant genotype, upregulated over time in the resistant genotype, hormone pathway genes, plant disease resistance genes, receptor-like kinases, contrasting expression profiles in QTL for disease resistance, and upregulated genes in enriched pathways. Overall, 22 candidate genes for SBS disease resistance were identified with support from the literature. These genes will be sources for future targeted mutagenesis and candidate gene validation potentially helping to improve disease resistance to this devastating foliar pathogen.

Lettuce Genotype-Dependent Effects of Temperature on Escherichia coli O157:H7 Persistence and Plant Head Growth

Lettuce has been commonly associated with the contamination of human pathogens, such as Escherichia coli O157:H7 (hereafter O157:H7), which has resulted in serious foodborne illnesses. Contamination events may happen throughout the farm-to-fork chain, when O157:H7 colonizes edible tissues and closely interacts with the plant. Environmental conditions have a significant impact on many plant–microbe interactions; however, it is currently unknown whether temperature affects O157:H7 colonization of the lettuce phyllosphere. In this study, we investigated the relationship between elevated growth temperatures, O157:H7 persistence, and lettuce head growth using 25 lettuce genotypes. Plants were grown under optimal or elevated temperatures for 3.5 weeks before being inoculated with O157:H7. The bacterial population size in the phyllosphere and lettuce head area was estimated at 0- and 10-days postinoculation (DPI) to assess bacterial persistence and head growth during contamination. We found that growing temperature can have a positive, negative, or no effect on O157:H7 persistence depending on the lettuce genotype. Furthermore, temperature had a greater effect on head area size than the presence of O157:H7. The results suggested that the combination of plant genotype and temperature level is an important factor for O157:H7 colonization of lettuce and the possibility to combine desirable food safety traits with heat tolerance into the lettuce germplasm.

A Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Didymella segeticola Causing Tea Leaf Spot.

Tea leaf spot caused by Didymella segeticola is an important disease that threatens the healthy growth of tea plants (Camellia sinensis) and results in reductions in the productivity and quality of tea leaves. Early diagnosis of the disease is particularly important for managing the infection. Loop-mediated isothermal amplification (LAMP) assay is an efficient diagnostic technique with the advantages of simplicity, specificity, and sensitivity. In this study, we developed a rapid, visual, and high-sensitivity LAMP assay for D. segeticola detection based on sequence-characterized amplified regions. Two pairs of amplification primers (external primers F3 and B3 and internal primers FIP and BIP) were designed based on a specific sequence in D. segeticola (NCBI accession number: OR987684). Compared to common pathogens of other genera in tea plants and other species in the Didymella genus (Didymella coffeae-arabicae, Didymella pomorum, and Didymella sinensis), the LAMP method is specific for detecting the species D. segeticola. The assay was able to detect D. segeticola at a minimal concentration of 1 fg/μL genomic DNA at an optimal reaction temperature of 65 °C for 60 min. When healthy leaves were inoculated with D. segeticola in the laboratory, the LAMP method successfully detected D. segeticola in diseased tea leaves at 72 h post inoculation. The LAMP assays were negative when the DNA samples were extracted from healthy leaves. Leaf tissues with necrotic lesions from 18 germplasms of tea plants tested positive for the pathogen by the LAMP assay. In summary, this study established a specific, sensitive, and simple LAMP method to detect D. segeticola, which provides reliable technical support for estimating disease prevalence and facilitates sustainable management of tea leaf spot.

Comparative Field Evaluation and Transcriptome Analysis Reveals that Chromosome Doubling Enhances Sheath Blight Resistance in Rice

Rice sheath blight, caused by Rhizoctonia solani Kihn (R. solani), poses a significant threat to rice production and quality. Autotetraploid rice, developed through chromosome doubling of diploid rice, holds great potential for enhancing biological and yield traits. However, its resistance to sheath blight in the field has remained unclear. In this study, the field resistance of 35 autotetraploid genotypes and corresponding diploids was evaluated across three environments from 2020 to 2021. The booting stage was optimal for inoculating period based on the inoculation and analysis of R. solani at five rice growth stages. We found autotetraploids generally exhibited lower disease scores than diploids, indicating enhanced resistance after chromosome doubling. Among the 35 genotypes, 16 (45.71%) displayed increased resistance, 2 (5.71%) showed decreased resistance, and 17 (48.57%) displayed unstable resistance in different sowing dates. All combinations of the genotype, environment and ploidy, including the genotype-environment-ploidy interaction, contributed significantly to field resistance. Chromosome doubling increased sheath blight resistance in most genotypes, but was also dependent on the genotype-environment interaction. To elucidate the enhanced resistance mechanism, RNA-seq revealed autotetraploid recruited more down-regulated differentially expressed genes (DEGs), additionally, more resistance-related DEGs, were down-regulated at 24 h post inoculation in autotetraploid versus diploid. The ubiquinone/terpenoid quinone and diterpenoid biosynthesis pathways may play key roles in ploidy-specific resistance mechanisms. In summary, our findings shed light on the understanding of sheath blight resistance mechanisms in autotetraploid rice.

Pathogenesis-Related 1 (PR1) Protein Family Genes Involved in Sugarcane Responses to Ustilago scitaminea Stress.

Plant resistance against biotic stressors is significantly influenced by pathogenesis-related 1 (PR1) proteins. This study examines the systematic identification and characterization of PR1 family genes in sugarcane (Saccharum spontaneum Np-X) and the transcript expression of selected genes in two sugarcane cultivars (ROC22 and Zhongtang3) in response to Ustilago scitaminea pathogen infection. A total of 18 SsnpPR1 genes were identified at the whole-genome level and further categorized into four groups. Notably, tandem and segmental duplication occurrences were detected in one and five SsnpPR1 gene pairs, respectively. The SsnpPR1 genes exhibited diverse physio-chemical attributes and variations in introns/exons and conserved motifs. Notably, four SsnpPR1 (SsnpPR1.02/05/09/19) proteins displayed a strong protein-protein interaction network. The transcript expression of three SsnpPR1 (SsnpPR1.04/06/09) genes was upregulated by 1.2-2.6 folds in the resistant cultivar (Zhongtang3) but downregulated in the susceptible cultivar (ROC22) across different time points as compared to the control in response to pathogen infection. Additionally, SsnpPR1.11 was specifically upregulated by 1.2-3.5 folds at 24-72 h post inoculation (hpi) in ROC22, suggesting that this gene may play an important negative regulatory role in defense responses to pathogen infection. The genetic improvement of sugarcane can be facilitated by our results, which also establish the basis for additional functional characterization of SsnpPR1 genes in response to pathogenic stress.

Transcriptome Analysis in Air-Liquid Interface Porcine Respiratory Epithelial Cell Cultures Reveals That the Betacoronavirus Porcine Encephalomyelitis Hemagglutinating Virus Induces a Robust Interferon Response to Infection.

Porcine hemagglutinating encephalomyelitis virus (PHEV) replicates in the upper respiratory tract and tonsils of pigs. Using an air-liquid interface porcine respiratory epithelial cells (ALI-PRECs) culture system, we demonstrated that PHEV disrupts respiratory epithelia homeostasis by impairing ciliary function and inducing antiviral, pro-inflammatory cytokine, and chemokine responses. This study explores the mechanisms driving early innate immune responses during PHEV infection through host transcriptome analysis. Total RNA was collected from ALI-PRECs at 24, 36, and 48 h post inoculation (hpi). RNA-seq analysis was performed using an Illumina Hiseq 600 to generate 100 bp paired-end reads. Differential gene expression was analyzed using DeSeq2. PHEV replicated actively in ALI-PRECs, causing cytopathic changes and progressive mucociliary disruption. Transcriptome analysis revealed downregulation of cilia-associated genes such as CILK1, DNAH11, LRRC-23, -49, and -51, and acidic sialomucin CD164L2. PHEV also activated antiviral signaling pathways, significantly increasing the expression of interferon-stimulated genes (RSAD2, MX1, IFIT, and ISG15) and chemokine genes (CCL5 and CXCL10), highlighting inflammatory regulation. This study contributes to elucidating the molecular mechanisms of the innate immune response to PHEV infection of the airway epithelium, emphasizing the critical roles of the mucociliary, interferon, and chemokine responses.

First Report of Bacterial Leaf Spot Disease on Sesame (Sesamum indicum L.) Caused by Pseudomonas syringae pv. sesami in North Carolina.

In July 2022, dark brown to black, angular, water-soaked lesions were observed on sesame leaves (Sesamum indicum L.) in a research plot established to assess yield potential for eight varieties at the North Carolina (NC) Sandhills Research Station (Chavez 2023). Symptoms were indicative of a bacterial leaf spot (BLS). At early flowering stage, leaf spots were present on scattered plants; varieties ES108, SS3301, and ES201 exhibited up to 75% disease prevalence, with lower frequency in ES103, S39, S4302, S3251, and S3276. Symptomatic leaves from 3-4 plants were collected on four different dates from July through September. A section of symptomatic tissue was excised and macerated in sterile deionized water (SDW). A 10 µL aliquot was streaked onto SPA medium (15 g sucrose, 5.0 g proteose peptone, 0.50 g MgSO4 7H2O, 0.25 g K2HPO4, 15 g agar per liter of SDW) and incubated at 28ºC. After 72 h, numerous, smooth, white-cream colored, convex-shaped, colonies were individually isolated. Five randomly selected isolates from the different collection dates, designated as AHP108-AHP111 and AHP116, were genotyped. The 16S rRNA, gyrB, rpoD, and gapA genes were sequenced (Heuer et al. 1997; Hwang et al. 2005) and deposited to NCBI (GenBank Accessions: P213467- PP213470; OQ628040-OQ628042; PP214983-PP214994; and PP255798). These five isolates shared 100% sequence identity for gyrB and rpoD. AHP108-AHP111 shared 100% sequence identity for 16S rRNA and gapA, with 99.7% and 90.8% identity, respectively, for AHP116. A phylogenetic tree was inferred from a maximum-likelihood analysis of concatenated gyrB, rpoD, and gapA sequences of the five isolates and the top 11 hts from a blastn search of the NCBI nucleotide database. Those hits included closely related sequences from Pseudomonas syringae pv. sesami type strains ICMP 763T and ICMP 7459T. Based on this phylogenetic analysis AHP108-AHP111 and AHP116 are P. syringae pv. sesami. Recent genomic analysis suggests this pathovar is part of P. amygdali (Gomila et al. 2017), but an official name change has not been proposed. Each of the five isolates were infiltrated into leaves of sesame varieties ES108, ES103, and S327, consistently resulting in similar symptoms. Thus, strain AHP116, as a representative, was used to fulfill Koch's postulates using five, 30-day-old potted sesame plants (var. S3301). Plants were spray-inoculated with a bacterial suspension of ~108 CFU/ml until runoff; plants were incubated in moist chambers 24 h pre and post inoculation at 28ºC with 80% relative humidity and a 12 h photoperiod. At 13 days post inoculation, symptoms resembling those on plants at the Sandhills Research Stations in 2022 were evident. Reisolated bacteria were confirmed to be AHP116 through 16S rRNA and gyrB amplification and sequencing. No symptoms were observed on the five water-inoculated plants. BLS of sesame has been reported in Asia and is thought to be seedborne (Firdous et al. 2009; Prathuangwong and Yowabutra 1997). To our knowledge, this is the first report of P. syringae pv. sesami causing BLS on sesame in North Carolina. Sesame cultivation in the state increased from approximately 2,000 acres in 2022 to 13,000 acres in 2023 and there is interest in cultivating sesame as a rotational and alternative crop because it requires minimal input costs. Potential outbreaks of BLS in this warm, humid region could negatively affect sesame production, where little is known about the economic impact of the disease.

A rapid strategy to develop personalized cancer nanovaccines for different immunogenic tumors.

e14670 Background: Therapeutic cancer vaccines aim to train the immune system and elicit antitumor immune responses, which are expected to fill the unmet medical need of immune checkpoint inhibitors. Despite tremendous efforts over the past decades, it remains challenging to prepare personalized cancer vaccines using a facile and rapid approach due to tumor diversity. CpG-ODN is a promising immunoadjuvant in cancer immunotherapy, and many clinical trials have been performed or are ongoing. However, commonly used phosphorothioate-modified CpG ODN easily induces protein aggregation and its long-term side effects are concerned. Here, we designed hollow large-pore mesoporous organosilica with sulfide bond to simultaneously load unmodified CpG-ODN with guanine-quadruplex (G4) structures and different tumor antigens to prepare personalized cancer vaccines in a simple, fast, and effective way. Methods: High-immunogenicE.G7-OVA lymphoma or low-immunogenic Lewis lung carcinoma (LLC) cells were subcutaneously inoculated into the left flanks to establish tumor-bearing mice. On days 4, 7, and 10 post tumor inoculation, cancer vaccines prepared using model antigen (OVA) or autologous tumor antigen (LLC tumor cell lysate) were subcutaneously injected into the right flanks of mice, respectively. Saline group, free tumor antigen group, free tumor antigen + CpG-ODN group, tumor antigen + organosilica group were used as controls. The size of tumors was monitored. Results: Cancer vaccines against E.G7-OVA showed high loading efficiencies of &gt;95% for both CpG-ODN and OVA, triggered antitumor immune response with high percentages of CD4+, CD8+, tetramer+CD8+ T cells in splenocytes, and effectively inhibited the growth of established tumor, compared with free OVA, free OVA + CpG-ODN, free OVA + organosilica groups. Cancer vaccines against LLC exhibited high loading efficiencies of &gt;95% and &gt;80% for CpG-ODN and LLC tumor cell lysate, activated antitumor immune response with high percentages of CD4+ and CD8+ T cells in splenocytes and CD8+ T, NK and M1 cells in tumor sites, and effectively inhibited the growth of established tumor, compared with control groups. Conclusions: Codelivery of CpG-ODN and different tumor antigens using hollow large-pore mesoporous organosilica elicits antitumor immunity against both high-immunogenic and low-immunogenic tumors.