A highly sensitive biotin-based probe for small RNA northern blot and its application in dissecting miRNA function in pepper.

Small RNAs play important roles in regulation of plant development and response to various stresses. Northern blot is an important technique in small RNA research. Isotope- and biotin- (or digoxigenin) labeled probes are frequently used in small RNA northern blot. However, isotope-based probe is limited by strict environmental regulation and availability in many places in the world while biotin-based probe is usually suffered from low sensitivity. In this study, we developed a T4 DNA polymerase-based method for incorporation of a cluster of 33 biotin-labeled C in small RNA probe (T4BC33 probe). T4BC33 probe reaches similar sensitivity as 32P-labeled probe in dot blot and small RNA northern blot experiments. Addition of locked nucleic acids in T4BC33 probe further enhanced its sensitivity in detecting low-abundance miRNAs. With newly developed northern blot method, expression of miR6027 and miR6149 family members was validated. Northern blot analysis also confirmed the successful application of virus-based miRNA silencing in pepper, knocking down accumulation of Can-miR6027a and Can-miR6149L. Importantly, further analysis showed that knocking-down Can-miR6027a led to upregulation of a nucleotide binding-leucine rich repeat domain protein coding gene (CaRLb1) and increased immunity against Phytophthora capsici in pepper leaves. Our study provided a highly sensitive and convenient method for sRNA research and identified new targets for genetic improvement of pepper immunity against P. capsici.

Multi-environment association study highlights candidate genes for robust agronomic quantitative trait loci in a novel worldwide Capsicum core collection.

Investigating crop diversity through genome-wide association studies (GWAS) on core collections helps in deciphering the genetic determinants of complex quantitative traits. Using the G2P-SOL project world collection of 10 038 wild and cultivated Capsicum accessions from 10 major genebanks, we assembled a core collection of 423 accessions representing the known genetic diversity. Since complex traits are often highly dependent upon environmental variables and genotype-by-environment (G × E) interactions, multi-environment GWAS with a 10 195-marker genotypic matrix were conducted on a highly diverse subset of 350 Capsicum annuum accessions, extensively phenotyped in up to six independent trials from five climatically differing countries. Environment-specific and multi-environment quantitative trait loci (QTLs) were detected for 23 diverse agronomic traits. We identified 97 candidate genes potentially implicated in 53 of the most robust and high-confidence QTLs for fruit flavor, color, size, and shape traits, and for plant productivity, vigor, and earliness traits. Investigating the genetic architecture of agronomic traits in this way will assist the development of genetic markers and pave the way for marker-assisted selection. The G2P-SOL pepper core collection will be available upon request as a unique and universal resource for further exploitation in future gene discovery and marker-assisted breeding efforts by the pepper community.

A 163-bp insertion in the Capana10g000198 encoding a MYB transcription factor causes male sterility in pepper (Capsicum annuum L.).

Male sterility provides an efficient approach for commercial exploitation of heterosis. Despite more than 20 genic male sterile (GMS) mutants documented in pepper (Capsicum annuum L.), only two causal genes have been successfully identified. Here, a novel spontaneous recessive GMS mutant, designated msc-3, is identified and characterized at both phenotypic and histological levels. Pollen abortion of msc-3 mutant may be due to the delayed tapetum degradation, leading to the non-degeneration of tetrads callosic wall. Then, a modified MutMap method and molecular marker linkage analysis were employed to fine mapping the msc-3 locus, which was delimited to the ~139.91-kb region harboring 10 annotated genes. Gene expression and structure variation analyses indicate the Capana10g000198, encoding a R2R3-MYB transcription factor, is the best candidate gene for the msc-3 locus. Expression profiling analysis shows the Capana10g000198 is an anther-specific gene, and a 163-bp insertion in the Capana10g000198 is highly correlated with the male sterile (MS) phenotype. Additionally, downregulation of Capana10g000198 in male fertile plants through virus-induced gene silencing resulted in male sterility. Finally, possible regulatory relationships of the msc-3 gene with the other two reported pepper GMS genes, msc-1 and msc-2, have been studied, and comparative transcriptome analysis reveals the expression of 16 GMS homologs are significantly downregulated in the MS anthers. Overall, our results reveal that Capana10g000198 is the causal gene underlying the msc-3 locus, providing important theoretical clues and basis for further in-depth study on the regulatory mechanisms of pollen development in pepper.

The responses of pepper plants to nitrogen form and dissolved oxygen concentration of nutrient solution in hydroponics.

BACKGROUND: The presence of oxygen in the growth medium is absolutely essential for root development and the overall metabolic processes of plants. When plants do not have an adequate oxygen supply for respiration, they can experience a condition known as hypoxia. In order to investigate the impact of different nitrogen forms and varying oxygen levels in nutrient solutions on the growth, photosynthesis, and chlorophyll fluorescence parameters of bell pepper plants, a comprehensive study was conducted. The experiment was designed as a factorial experiment, considering two main factors: nitrogen forms (calcium nitrate and ammonium sulfate) with a fixed nitrogen concentration of 5 mM, and the oxygen levels of the nutrient solutions (ranging from 1.8 ± 0.2 to 5.3 ± 0.2 mg. L-1). RESULTS: The study examined the effects of nitrogen (NH4+ and NO3-) application on various parameters of vegetative growth. The results demonstrated that the use of ammonium (NH4+) led to a reduction in the most measured parameters, including the fresh and dry mass of both the root and shoot, at low O2 concentrations of 1.8 ± 0.2; 2.6 ± 0.2 and 3.8 ± 0.2 mg. L-1. However, an interesting observation was made regarding the impact of oxygen levels on root growth in plants grown with nitrate (NO3-). Specifically, the highest levels of oxygen significantly increased root growth in NO3--fed plants. Additionally, the application of NH4+ resulted in an increase in chlorophyll concentration in the leaves, particularly when combined with high oxygen levels in the nutrient solution. On the other hand, leaves of plants fed with NO3- exhibited higher photosynthetic rate (A), intrinsic water use efficiency (iWUE), and instantaneous carboxylation efficiency (A/Ci) compared to those fed with NH4+. Furthermore, it was found that NO3--fed plants displayed the highest instantaneous carboxylation efficiency at oxygen levels of 3.8 and 5.3 mg. L-1, while the lowest efficiency was observed at oxygen levels of 1.8 and 2.6 mg. L-1. In contrast, NH4+-grown plants exhibited a higher maximal quantum yield of PSII photochemistry (Fv/Fm), as well as increased variable fluorescence (Fv) and maximum fluorescence (Fm), compared to NO3--grown plants. Interestingly, the NO3--fed plants showed an increase in Fv/Fm, Fv, and Fm with the elevation of oxygen concentration in the nutrient solution up to 5.3 mg. L-1. CONCLUSION: This study showed that, the growth and photosynthesis parameters in bell pepper plants are sensitive to oxygen stress in floating hydroponic culture. Therefore, the oxygen level in the nutrient solution must not be lower than 3.8 and 5.3 mg. L-1 in NH4+ and NO3- -supplied culture media or nutrient solutions, respectively.

Differential responses of chili varieties grown under cadmium stress.

Heavy metal cadmium (Cd) naturally occurs in soil and is a hazardous trace contaminant for humans, animals, and plants. The main sources of Cd pollution in soil include overuse of phosphatic fertilizers, manure, sewage sludge, and aerial deposition. That's why an experiment was conducted to analyze the effect of Cd toxicity in Capsicum annuum L. by selecting its seven varieties: Hybrid, Desi, Sathra, G-916, BR-763, BG-912, and F1-9226. Cadmium was spiked in soil with four levels, i.e., (0, 3, 4, and 5 mg Cd kg- 1 of soil) for a week for homogeneous dispersion of heavy metal. Chili seeds were sown in compost-filled loamy soil, and 25-day-old seedlings were transplanted into Cd-spiked soil. Cadmium increasing concentration in soil decreased chili growth characteristics, total soluble sugars, total proteins, and amino acids. On the other hand, the activities of antioxidant enzymes were increased with the increasing concentration of Cd in almost all the varieties. Treatment 5 mg Cd/kg application caused - 197.39%, -138.78%, -60.77%, -17.84%, -16.34%, -11.82% and - 10.37% decrease of carotenoids level in chili V2 (Desi) followed by V4 (G-916), V1 (Hy7brid), V7 (F1-9226), V6 (BG-912), V5 (BR-763) and V3 (Sathra) as compared to their controls. The maximum flavonoids among varieties were in V5 (BR-763), followed by V6 (BG-912), V7 (F1-9226), V3 (Sathra) and V1 (Hybrid). Flavonoids content was decreased with - 37.63% (Sathra), -34.78% (Hybrid), -33.85% (G-916), -31.96% (F1-9226), -31.44% (Desi), -30.58% (BR-763), -22.88% (BG-912) as compared to their control at 5 mg Cd/kg soil stress. The maximum decrease in POD, SOD, and CAT was - 31.81%, -25.98%, -16.39% in chili variety V7 (F1-9226) at 5 mg Cd/kg stress compared to its control. At the same time, maximum APX content decrease was - 82.91%, followed by -80.16%, -65.19%, -40.31%, -30.14%, -10.34% and - 6.45% in V4 (G-916), V2 (Desi), V3 (Sathra), V6 (BG-912), V1 (Hybrid), V7 (F1-9226) and V5 (BR-763) at 5 mg Cd/kg treatment as compared to control chili plants. The highest CAT was found in 5 chili varieties except Desi and G-916. Desi and G-916 varieties. V5 (BR-763) and V6 (BG-912) were susceptible, while V1 (Hybrid), V3 (Sathra), and V7 (F1-9226) were with intermediate growth attributes against Cd stress. Our results suggest that Desi and G-916 chili varieties are Cd tolerant and can be grown on a large scale to mitigate Cd stress naturally.

Elucidating the callus-to-shoot-forming mechanism in Capsicum annuum 'Dempsey' through comparative transcriptome analyses.

BACKGROUND: The formation of shoots plays a pivotal role in plant organogenesis and productivity. Despite its significance, the underlying molecular mechanism of de novo regeneration has not been extensively elucidated in Capsicum annuum 'Dempsey', a bell pepper cultivar. To address this, we performed a comparative transcriptome analysis focusing on the differential expression in C. annuum 'Dempsey' shoot, callus, and leaf tissue. We further investigated phytohormone-related biological processes and their interacting genes in the C. annuum 'Dempsey' transcriptome based on comparative transcriptomic analysis across five species. RESULTS: We provided a comprehensive view of the gene networks regulating shoot formation on the callus, revealing a strong involvement of hypoxia responses and oxidative stress. Our comparative transcriptome analysis revealed a significant conservation in the increase of gene expression patterns related to auxin and defense mechanisms in both callus and shoot tissues. Consequently, hypoxia response and defense mechanism emerged as critical regulators in callus and shoot formation in C. annuum 'Dempsey'. Current transcriptome data also indicated a substantial decline in gene expression linked to photosynthesis within regenerative tissues, implying a deactivation of the regulatory system governing photosynthesis in C. annuum 'Dempsey'. CONCLUSION: Coupled with defense mechanisms, we thus considered spatial redistribution of auxin to play a critical role in the shoot morphogenesis via primordia outgrowth. Our findings shed light on shoot formation mechanisms in C. annuum 'Dempsey' explants, important information for regeneration programs, and have broader implications for precise molecular breeding in recalcitrant crops.

CaIAA2-CaARF9 module mediates the trade-off between pepper growth and immunity.

To challenge the invasion of various pathogens, plants re-direct their resources from plant growth to an innate immune defence system. However, the underlying mechanism that coordinates the induction of the host immune response and the suppression of plant growth remains unclear. Here we demonstrate that an auxin response factor, CaARF9, has dual roles in enhancing the immune resistance to Ralstonia solanacearum infection and in retarding plant growth by repressing the expression of its target genes as exemplified by Casmc4, CaLBD37, CaAPK1b and CaRROP1. The expression of these target genes not only stimulates plant growth but also negatively impacts pepper resistance to R. solanacearum. Under normal conditions, the expression of Casmc4, CaLBD37, CaAPK1b and CaRROP1 is active when promoter-bound CaARF9 is complexed with CaIAA2. Under R. solanacearum infection, however, degradation of CaIAA2 is triggered by SA and JA-mediated signalling defence by the ubiquitin-proteasome system, which enables CaARF9 in the absence of CaIAA2 to repress the expression of Casmc4, CaLBD37, CaAPK1b and CaRROP1 and, in turn, impeding plant growth while facilitating plant defence to R. solanacearum infection. Our findings uncover an exquisite mechanism underlying the trade-off between plant growth and immunity mediated by the transcriptional repressor CaARF9 and its deactivation when complexed with CaIAA2.

Carotenoid retention during post-harvest storage of Capsicum annuum: the role of the fruit surface structure.

In this study, a chilli pepper (Capsicum annuum) panel for post-harvest carotenoid retention was studied to elucidate underlying mechanisms associated with this commercial trait of interest. Following drying and storage, some lines within the panel had an increase in carotenoids approaching 50% compared with the initial content at the fresh fruit stage. Other lines displayed a 25% loss of carotenoids. The quantitative determination of carotenoid pigments with concurrent cellular analysis indicated that in most cases, pepper fruit with thicker (up to 4-fold) lipid exocarp layers and smooth surfaces exhibit improved carotenoid retention properties. Total cutin monomer content increased in medium/high carotenoid retention fruits and subepidermal cutin deposits were responsible for the difference in exocarp thickness. Cutin biosynthesis and cuticle precursor transport genes were differentially expressed between medium/high and low carotenoid retention genotypes, and this supports the hypothesis that the fruit cuticle can contribute to carotenoid retention. Enzymatic degradation of the cuticle and cell wall suggests that in Capsicum the carotenoids (capsanthin and its esters) are embedded in the lipidic exocarp layer. This was not the case in tomato. Collectively, the data suggest that the fruit cuticle could provide an exploitable resource for the enhancement of fruit quality.

Temperature-dependent action of pepper mildew resistance locus O 1 in inducing pathogen immunity and thermotolerance.

Plant diseases tend to be more serious under conditions of high-temperature/high-humidity (HTHH) than under moderate conditions, and hence disease resistance under HTHH is an important determinant for plant survival. However, how plants cope with diseases under HTHH remains poorly understood. In this study, we used the pathosystem consisting of pepper (Capsicum annuum) and Ralstonia solanacearum (bacterial wilt) as a model to examine the functions of the protein mildew resistance locus O 1 (CaMLO1) and U-box domain-containing protein 21 (CaPUB21) under conditions of 80% humidity and either 28 °C or 37 °C. Expression profiling, loss- and gain-of-function assays involving virus-induced gene-silencing and overexpression in pepper plants, and protein-protein interaction assays were conducted, and the results showed that CaMLO1 acted negatively in pepper immunity against R. solanacearum at 28 °C but positively at 37 °C. In contrast, CaPUB21 acted positively in immunity at 28 °C but negatively at 37 °C. Importantly, CaPUB21 interacted with CaMLO1 under all of the tested conditions, but only the interaction in response to R. solanacearum at 37 °C or to exposure to 37 °C alone led to CaMLO1 degradation, thereby turning off defence responses against R. solanacearum at 37 °C and under high-temperature stress to conserve resources. Thus, we show that CaMLO1 and CaPUB21 interact with each other and function distinctly in pepper immunity against R. solanacearum in an environment-dependent manner.

Amphistomy: stomata patterning inferred from 13C content and leaf-side-specific deposition of epicuticular wax.

BACKGROUND AND AIMS: The benefits and costs of amphistomy (AS) vs. hypostomy (HS) are not fully understood. Here, we quantify benefits of access of CO2 through stomata on the upper (adaxial) leaf surface, using 13C abundance in the adaxial and abaxial epicuticular wax. Additionally, a relationship between the distribution of stomata and epicuticular wax on the opposite leaf sides is studied. METHODS: We suggest that the 13C content of long-chain aliphatic compounds of cuticular wax records the leaf internal CO2 concentration in chloroplasts adjacent to the adaxial and abaxial epidermes. This unique property stems from: (1) wax synthesis being located exclusively in epidermal cells; and (2) ongoing wax renewal over the whole leaf lifespan. Compound-specific and bulk wax 13C abundance (δ) was related to amphistomy level (ASL; as a fraction of adaxial in all stomata) of four AS and five HS species grown under various levels of irradiance. The isotopic polarity of epicuticular wax, i.e. the difference in abaxial and adaxial δ (δab - δad), was used to calculate the leaf dorsiventral CO2 gradient. Leaf-side-specific epicuticular wax deposition (amphiwaxy level) was estimated and related to ASL. KEY RESULTS: In HS species, the CO2 concentration in the adaxial epidermis was lower than in the abaxial one, independently of light conditions. In AS leaves grown in high-light and low-light conditions, the isotopic polarity and CO2 gradient varied in parallel with ASL. The AS leaves grown in high-light conditions increased ASL compared with low light, and δab - δad approached near-zero values. Changes in ASL occurred concomitantly with changes in amphiwaxy level. CONCLUSIONS: Leaf wax isotopic polarity is a newly identified leaf trait, distinguishing between hypo- and amphistomatous species and indicating that increased ASL in sun-exposed AS leaves reduces the CO2 gradient across the leaf mesophyll. Stomata and epicuticular wax deposition follow similar leaf-side patterning.

Effect of a Zinc Phosphate Shell on the Uptake and Translocation of Foliarly Applied ZnO Nanoparticles in Pepper Plants (Capsicum annuum).

Here, isotopically labeled 68ZnO NPs (ZnO NPs) and 68ZnO NPs with a thin 68Zn3(PO4)2 shell (ZnO_Ph NPs) were foliarly applied (40 µg Zn) to pepper plants (Capsicum annuum) to determine the effect of surface chemistry of ZnO NPs on the Zn uptake and systemic translocation to plant organs over 6 weeks. Despite similar dissolution of both Zn-based NPs after 3 weeks, the Zn3(PO4)2 shell on ZnO_Ph NPs (48 ± 12 nm; -18.1 ± 0.6 mV) enabled a leaf uptake of 2.31 ± 0.34 µg of Zn, which is 2.7 times higher than the 0.86 ± 0.18 µg of Zn observed for ZnO NPs (26 ± 8 nm; 14.6 ± 0.4 mV). Further, ZnO_Ph NPs led to higher Zn mobility and phloem loading, while Zn from ZnO NPs was stored in the epidermal tissues, possibly through cell wall immobilization as a storage strategy. These differences led to higher translocation of Zn from the ZnO_Ph NPs within all plant compartments. ZnO_Ph NPs were also more persistent as NPs in the exposed leaf and in the plant stem over time. As a result, the treatment of ZnO_Ph NPs induced significantly higher Zn transport to the fruit than ZnO NPs. As determined by spICP-TOFMS, Zn in the fruit was not in the NP form. These results suggest that the Zn3(PO4)2 shell on ZnO NPs can help promote the transport of Zn to pepper fruits when foliarly applied. This work provides insight into the role of Zn3(PO4)2 on the surface of ZnO NPs in foliar uptake and in planta biodistribution for improving Zn delivery to edible plant parts and ultimately improving the Zn content in food for human consumption.

Unveiling the Biomedical Applications of Novel Coumarins Isolated From Capsicum Annuum L. Seeds by a Multivariate Extraction Technique.

For the first time, kinetic thermomagnetic extraction is a novel approach presented in this work. It required the application of four distinct variables: rotation speed (50, 75, and 100 rpm), magnetic field (0.8, 1.2, and 1.6 T), time interval (30, 60, and 90 min), and temperature (45, 55, and 65 °C). Numerous phytochemical categories were detected in the 81 crude chloroform extracts of green sweet bell pepper seeds that were collected, according to phytochemical analysis. Nine extracts were discovered to be linked to the coumarin chemical class and to have the same two extraction parameters: a 90-minute extraction duration and a 55 °C extraction temperature. To enable their coumarin contents to be chemically separated and chromatographically purified, two of these extracts containing coumarin were chosen. Four new phytocoumarins have been identified and their molecular structures distinguished using FTIR spectra, 1H-NMR, 13C-NMR, and mass analysis. By using MTT probing, it was discovered that these phytocoumarins exhibited anticancer activities against eight malignant populations and reduced oxidative stress in human SH-SY5Y populations. Similarly, the anti-inflammatory and antidiabetic properties were determined using three and two associated enzymes, respectively. The results demonstrated that the extracted phytocoumarins have exceptional oxidative stress-mitigating characteristics, ranging from 71.51 to 81.48 %, when compared to a positive control. Furthermore, they showed excellent cytotoxicity against the test malignant populations (IC50 values of 46.76-81.45 µg/ml). The isolates need to be taken into account as dual COX-2/5-LOX antagonists because they also showed a fascinating selective anti-inflammatory effect. The phytocoumarins under investigation have selectivity indices that are higher than those of the standards used, suggesting that they may have the ability to selectively block the COX2 enzyme that induces harmful inflammation. Compared to the standards, the phytocoumarins have a higher ability to block the catalytic activity of 5-LOX. This observation suggests that the phytocoumarins are powerful 5-LOX agents. Finally, they had a modest antidiabetic impact when tested against two blood-controlling enzymes. The authors came to the conclusion that the technique adopted is flexible and successful for extraction after modifying its components. Moreover, isolated phytocoumarins in general and natural-B1 in particular provide naturally derived solutions for oxidative stress and its associated diseases.

First Report of Fusarium falciforme Causing Fusarium Wilt on pepper in Hainan, China.

Pepper (Capsicum annuum L.) is a significant vegetable crop, valued for its nutritional and economic importance (Pang et al. 2023). Pepper cultivation in China accounts for about 8-10% of the total vegetable planting area, contributing an output value of approximately 250 billion yuan. This makes pepper the leading vegetable in terms of both planting area and economic value. In December 2023, a total of 70% disease incidence of Fusarium wilt was observed in a 1200 m² pepper seed breeding base in Sanya City, Hainan Province, China (18°38'60″ N, 109°16'51″ E). Symptoms initially appeared as wilting on upper leaves. Subsequently, the base of the stem started to necrosis, browning, and gradually spreading upward along the stem. As the lesions expanded, the whole plant gradually wilted and died. Ten diseased plants were randomly selected from the most severely affected area (667 m²). Diseased tissues (5 mm²) were subsequently removed from the lesion edges of these plants, surface sterilized in 75% ethanol for 30 s, and rinsed with sterile distilled water three times, finally cultured on potato dextrose agar (PDA) at 25 °C. Six fungal isolates were obtained using the single-spore isolation method (HN-01 to HN-06). Colonies produced white aerial mycelia with apricot pigments in the PDA medium. The spore morphology and size were observed and measured using synthetic nutrient-poor agar (SNA) medium. Macroconidia were hyaline, slightly curved in shape with 3 or 4 septa, measuring 28.6 to 41.4 × 3.2 to 6.2 µm (av. = 34.8 ± 3.32 × 4.6 ± 0.85 um, n = 20). Microconidia were elongated, oval with 0 or 1 septum, and measured 11.2 to 16.8 × 2.6 to 5.8 µm (av. = 13.5 ± 1.47 × 4.12 ± 1.03 um, n = 20). Chlamydospores were spherical, terminal or intercalary, solitary or chain-forming, with diameters ranging from 2.8 to 10.5 um (av. = 5.8 ± 2.31 um, n = 20). For molecular identification, genomic DNA from all six isolates was extracted using the cetyl trimethyl ammonium bromide (CTAB) method, and the internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (EF1-α), and RNA polymerase II beta subunit (RPB2) regions were amplified and sequenced using the primers ITS1/ITS4, EF-1/EF-2, and RPB2-5F/7cR (White et al. 1990; O'Donnell et al. 2010). The sequences were deposited in GenBank (ITS: PP779839, PP779840, PP779841, PP779842, PP779843, PP779844; EF1-α: PP797138, PP797139, PP797140, PP797141, PP797142, PP797143; RPB2: PP797144, PP797145, PP797146, PP797147, PP797148, PP797149). The sequences of all three genes showed 99 to 100% similarity with Fusarium falciforme and other closely related Fusarium species (ITS: PP735125, EF1-α: OP163897 and RPB2: MF467484). A maximum likelihood phylogenetic tree was constructed based on the ITS, EF1-α, and RPB2 sequences of all isolates, along with other closely related Fusarium species. Based on morphological and phylogenetic characteristics, all isolates were identified as F. falciforme (Xu et al. 2023; Wang et al. 2023). Ten Pepper inbred line A23-41 (5 for the inoculation treatment and 5 for the control) were tested for pathogenicity using three representative isolates: HN-01, HN-02, and HN-03. A total of 20 ul of spore suspension with concentration 106 spores/ml was injected near the soil surface of the stem, while the control treatment was inoculated with 20 µl of sterile water. The plants were placed in a climatic chamber at RH80-90% and 25/20 °C (day/night) after inoculation. The experiment was repeated three times. After 10 days, Inoculated plants stem developed necrosis, browning, while the control group remained asymptomatic. The fungus reisolated from the artificially infected stems was identified through EF1-α and RPB2 sequence analysis as F. falciforme, thus fulfilling Koch's postulates. Fusarium falciforme has been previoulsy reported as causing various diseases on different hosts in several countries, including Korea (Kang et al., 2024), Malaysia (Balasubramaniam et al., 2023), and Mexico (Payán-Arzapalo et al., 2024). To the best of our knowledge, this is the initial report demonstrating that F. falciforme causes Fusarium wilt on peppers in China. The study results can provide the basis for future research on the occurrence, prevention, and management of this disease.

First Report of Colletotrichum gigasporum causing Anthracnose of chili pepper in South Korea.

Anthracnose, a destructive fungal disease, poses a significant threat to chili pepper (Capsicum annuum L.) production worldwide (de Silva et al. 2019). In South Korea, anthracnose outbreaks have traditionally been attributed to several Colletotrichum species such as C. gloeosporioides and C. acutatum. About 10% of the yield (chili production) is lost annually in South Korea due to chili anthracnose (Oo et al. 2020). During field surveys conducted in August 2017, symptomatic lesions resembling anthracnose were observed on chili pepper in two farmer's fields (Gochang and Cheongyang) in South Korea. Affected fruits exhibited characteristic symptoms, including circular sunken lesions with dark margins and abundant orange spore masses on the surface. About 20% of chili pepper fruit were affected in each field with an area of about 0.2 ha. Five putative Colletotrichum spp. isolates were obtained from six affected fruits (three from each field) following the procedure described by Cai et el. (2009). Three isolates (C01049, C01111, and C01115), representing each location, were selected to identify at the species level. Colonies on potato dextrose agar (incubated at 25°C in the dark for 7 days) were cottony with entire margins, white aerial mycelium and dark gray in the center. Conidia were hyaline, aseptate, cylindrical with bothnds round, and 17.8 - 30.5 × 6.0 -10.0 µm (mean 23.8 ×7.9 µm, n = 30). Appressoria were dark brown, irregular but mostly ovoid with smooth walls. These morphological features align with those of Colletotrichum spp. within the Colletotrichum gigasporum species (Liu et al. 2014). The identity of the pathogen was further confirmed through multi-locus phylogenetic analysis. The target genes including ITS, ACT, CHS-1, GAPDH, TUB2, and GS were amplified and sequenced using the primer sets ITS1/ITS4, ACT 512F/ ACT-783R, CHS-79F/ CHS-345R, GDF/GDR, T1/Bt2b, and GSF1/GSR1, respectively (Weir et al. 2012; Liu et al. 2014). The resulting sequences were deposited in GenBank (accession no: ITS: MT605261, MT605262, LC823714; ACT: MT612991, MT612992, LC823718; CHS-1: MT612993, MT612994, LC823717; GAPDH: LC811375, LC811376, LC823716; TUB2: MT612997, MT612998, LC823715; GS: LC811377, LC811378, LC823719). The constructed Bayesian and maximum likelihood tree based on combined sequences of ITS, ACT, CHS-1, GAPDH, TUB2, and GS confirmed the identification of the isolates (C01049, C01111, C01115) as C. gigasporum. Pathogenicity tests were conducted by inoculating healthy chili fruit with 70 µL of a conidial suspension (1×106 conidia /mL) of pure cultures of the isolates. The conidial suspension was applied on 10 wounded or 10 non-wounded fruit. The same number of fruit were treated with sterile distilled water as controls. Within 5 days of inoculation, symptoms consistent with anthracnose developed on the inoculated wounded fruit, whereas non-wounded and control fruit remained asymptomatic. This experiment was repeated twice. Colletotrichum gigasporum was re-isolated from diseased tissue of inoculated fruit. Colletotrichum gigasporum has been identified as the cause of anthracnose on Dalbergia odorifera, Carica papaya in China, and Brassica oleracea in India (Wan et al., 2018; Saini et al. 2022; He et al. 2023). To the best of our knowledge, this report marks the first documented instance of C. gigasporum causing anthracnose of chili pepper in South Korea. These results indicate that various species of Colletotrichum can be the fungi causing chili pepper anthracnose. The findings of this study emphasize the need for effective disease management strategies to mitigate impact of C. gigasporum on chili pepper cultivation in the region.

First report of Colletotrichum queenslandicum associated with chilli anthracnose in Mauritius.

Globally, chilli (Capsicum annuum L.) is one of the most economically important and widely cultivated crop which elicits ethnomedicinal and nutritional potential as well as enhancing the taste and aroma of foods (Ayob et al., 2022; Kiran et al., 2020). Anthracnose disease is regarded as a prime constraint in chilli production, leading to enormous losses in tropical and subtropical countries. In September 2022, chilli fruit displaying sunken, shriveled and dark bown to black lesion with abundant acervuli on the surface was obtained from Flacq, Mauritius. From the symptomatic tissue, small pieces of the diseased tissue were excised, surface-disinfected using 1% sodium hypochlorite, twice rinsed using sterilized distilled water, air-dried and plated on PDA. After 7 days of incubation at room temperature, white to greyish white colony with dense white cottony aerial mycelium was recovered. Out of two isolates, CHF and CH10, the latter was considered for morphological and molecular characterization. The observed conidia (n=30) were unicellular, straight, cylindrical with rounded ends and slight constriction near the centre and had average length and width of 20.5 µm and 6 µm, respectively. For growth rate measurement of the isolate, two 5×5 mm of fungal agar plugs were taken from growing edge of colony, inoculated at centre of individual PDA plate and incubated at room temperature with a natural light/dark cycle. The diameter of the cultures were measured perpendicularly for a period of 7 days and the growth rate was calculated as 7-day average of daily growth (mm day-1). The growth rate of the fungal isolate (CH10) was 13.5 mm day-1 on PDA. Based on the morphological characters, the isolate was classified within the C. gloeosporioides species complex. For precise identification of the isolate, DNA was extracted from fungal mycelium using traditional DNA isolation methods (Ranghoo and Hyde, 2000), followed by PCR amplification and DNA sequencing using primer pairs ITS4/ITS5 (White et al., 1990), GDF/GDR and T1/Bt2b (Gan et al., 2016), respectively. ITS gene sequence (600 bp) confirmed that the isolate was Colletotrichum, with 99.83% similarity to KR704204 while GADPH (277 bp), TUB2 (733 bp) and ApMat (801 bp) gene sequences showed 99.64 to 100% similarity to C. queenslandicum with GenBank reference sequences, KT372374, KU221378 and MG674932 respectively. The gene sequences of isolate CH10 were deposited in GenBank database under the following accession numbers OR681557 (ITS), OR233734 (GADPH), OR475575 (TUB2) and PP622748 (ApMat). Koch's postulates were confirmed by spraying disease-free chilli plants with 10µL of conidial suspension (1 × 106 spores/ml) prepared from 7 days old colony of isolate CH10. Healthy chilli plants inoculated with sterile distilled water served as a negative control experiment. The plants were grown in pots in a moist chamber at 25˚C. After 5 days post-inoculation, anthracnose symptoms were developed on test plants while the control plant remained asymptomatic. The original isolate was successfully recovered from the test fruits, thus fulfilling Koch's postulates. The experiment was repeated thrice and revealed the same results. To the best of our knowledge, this is the first record of C. queenslandicum in Mauritius and is the first time to report anthracnose of chilli caused by this fungus. Colletotrichum queenslandicum has previously been reported in Europe, Mexico, US, Puerto Rico, Australia, Fiji, Brazil, Indonesia and China. Furthermore, the latter was associated with papaya, avocado, cashew, coffee, Persian lime, Licania tomentosa, white mangrove, lychee, mango, Nephelium lappaceum, olive, passionfruit, Dracaena cambodiana and Syzygium australe (Câmara and Vieira, 2022; Shidiq et al., 2024; Wang et al., 2022). This study will allow local farmers training and extension facilities to increase awareness among farmers about this disease-causing agent and allow them to take necessary measures for building up chilli crops resilience against this new and emerging pathogen in Mauritius.

First Report of Paprika mild mottle virus Infecting pepper (Capsicum annuum L.) in China.

Paprika mild mottle virus (PaMMV), a tobamovirus from the Virgaviridae family, has been reported to infect pepper (Capsicum annuum L.) in the Netherlands (García-Luque et al., 1993), Japan (Hamada et al., 2003), Bulgaria (Ruíz del Pino et al., 2003), and Israel (Luria et al., 2018). Pepper has become the most widely planted vegetable and the most heavily consumed spicy condiment, with a large planting area (>21,000 km2) in China (Zou et al., 2021). However, the crop's productivity is limited by viral diseases. In August 2021, a survey of viral diseases was conducted across five pepper-growing regions in Linyi, Shandong Province, China, revealed that 5% to 10% of plants in most fields exhibited symptoms such as stunting, leaf narrowing, chlorosis, and crinkling of leaf tissue (Fig. S1-a). Subsequently, seven symptomatic samples (Fig. S1-b) were collected from a plot (118° 29' E, 34° 65' N) where thin-skinned peppers were grown. All samples tested positive in a Western blot assay using a polyclonal antibody for tobacco mosaic virus (TMV), indicating the presence of a tobamovirus (Fig. S1-c). To identify the virus, total RNAs were extracted from 10 different symptomatic pepper plant samples, using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) and combined into a single sample in equal amounts (100 ng/µL each). RNA-Seq was performed on this mixture using the Illumina NovaSeq 6000 (Illumina, San Diego, USA). Raw reads (6.92G) in fastq format were processed with in-house perl scripts to yield clean reads (6.68G), which were assembled into 780 contigs (mean length: 2036 bp) using Trinity software (v2.6.6). These contigs were then analysed using the NCBI BLASTX program (http://www.ncbi.nlm.nih.gov/blast) against the viral RefSeq database. The results demonstrated that the contigs exhibited an average genome coverage of 40.16%. Notably, one unique contig (6453 bp) was mapped to the PaMMV genome (reference sequence KX187305.1) with 98.34% identity. To verify the RNA-Seq result, a PaMMV-specific primer pair (forward: 5'-GAGTTCATAGAGGCAGTACC-3'; reverse: 5'-CTTCGATTTAAGTGGAGGGAT-3') was designed for amplifying an 800-nt fragment of the PaMMV coat protein (CP) gene by RT-PCR. The 800-nt fragment was successfully amplified from all the symptomatic samples (Fig. S1-d). Sequenced RT-PCR products (GenBank No. OR365081.1) showed 99.75% nucleotide identity with PaMMV isolates (OQ198318.1, China, goat) according to BLAST analysis. Mechanical transmission of PaMMV from three infected pepper plants collected in Linyi to healthy pepper plants confirmed pathogenicity. Three pepper cultivars (trade name: Qiemen, Tianyu, Haonong11) were tested and showed varying symptoms post-inoculation (Fig. S1-e). All cultivars were confirmed in the lab to be PMMoV-sensitive and exhibited significant viral disease symptoms after PMMoV inoculation (data not shown). Each variety was inoculated with at least 3 seedlings from different field-collected virulence sources. Qiemen (cultivar: sweet pepper) exhibited severe symptoms with necrotic spots, Tianyu (cultivar: Chaotian pepper) had mild leaf wrinkling, and Haonong11 (cultivar: Pickled pepper) showed yellow mottle and leaf shrinking. All tested seedlings were positive for PaMMV by RT-PCR (Fig. S1-f). To our knowledge, this is the first report of PaMMV infecting pepper in China, highlighting the need for vigilant monitoring to protect the chili industry and prevent the virus's spread to other crops.

First Report of Xanthomonas perforans Causing Bacterial Spot of Pepper (Capsicum annuum) in North Carolina.

North Carolina (NC) is the fifth largest producer of bell pepper (Capsicum annuum) in the US with an estimated 2,400 acres in production (NASS-USDA, 2022). A survey of bacterial diseases of peppers was initiated in 2020 after numerous bacterial spot outbreaks were reported in NC. Bacterial spot is caused by a complex of four Xanthomonads: X. euvesicatoria, X. vesicatoria, X. perforans, and X. hortorum pv. gardneri (Larrahondo-Rodríguez et al., 2022). If not preemptively managed, bacterial spot can cause up to 40% yield loss (Kousik and Ritchie, 1998). During the 2020 and 2021 growing seasons, 103 yellow mucoid colonies were isolated from plants representing 51 pepper cultivars symptomatic of bacterial spot, i.e., water-soaked leaf lesions that become necrotic spots on leaves and fruits across 22 commercial fields in NC following published methods (Klein-Gordon et al., 2021). All colonies were characterized to species using the qPCR species-specific primers and probes described by Strayer et al. 2016. Of the 103 colonies, 12 isolates tested positive for X. perforans. To confirm qPCR results, a Multi-Locus Sequence Analysis (MLSA) was run using fusA, gapA, gltA, gyrB, and lacF following previously described methods (Almeida et al., 2010) on three representative isolates: AHX61, collected in September 2020 from a field with 20% disease severity in Wake County on cv. Canary Bell; AHX261, collected in July 2021 from a field with 50% disease severity in Sampson County on Jalapeño; and AHX426, collected in August 2021 from a field with 50% disease severity in Dublin County on Jalapeño. All gene sequences were deposited to NCBI (GenBank Accessions: OQ799538-OQ799552) and compared to those from X. euvesicatoria, X. hortorum pv. gardneri, X. perforans, and X. vesicatoria type strains (Almeida et al., 2010). The MLSA showed AHX61, AHX261, and AHX426 cluster with X. perforans ICMP16690T, sharing 99-100% nucleotide similarity. Koch's postulates were performed with the three strains, Xp1484T [ X. perforans type strain, (Wilson 1987)], and water as a negative control. Three 10-week-old bell pepper plants (cv. Early Cal Wonder) were dip-inoculated in 600 mL of a bacterial suspension at an OD600 of 0.3 (~5x108 CFU/mL) and 0.04% Silwet L-77 per strain or water. All 18 plants were individually incubated in a plastic bag for 48 h post-inoculation at 28°C, 80% relative humidity, and 14 h:10 h light-dark cycle in a growth chamber, after which plastic bags were removed. Water-soaking and necrotic spots characteristic of bacterial spot were first observed at six days post-inoculation (dpi). At 14 dpi, symptomatic leaves were removed from treated plants to attempt pathogen re-isolation. Yellow mucoid colonies similar in morphology to those originally inoculated were recovered from all plants and confirmed to be X. perforans through sequencing; no isolates were recovered from water-treated plants. To our knowledge, this is the first time X. perforans is isolated in commercial bell pepper and specialty pepper fields in the state. This is an indication that the Xanthomonas population on peppers in the state is more diverse than previously reported and that pathogen populations will require monitoring for possible species shifts for this crop in NC.

Members of the Capsicum annuum CaTrxh Family Respond to High Temperature and Exhibit Dynamic Hetero/Homo Interactions.

Climate change adversely affects the water and temperature conditions required for plant growth, leading to a decrease in yield. In high temperatures, oxidative stress causes cellular damage in plant cells, which is a negative factor for crop production. Thioredoxin (Trx) is a small redox protein containing a conserved WC(G/P)PC motif that catalyzes the exchange of disulfide bonds. It is known to play an important role in maintaining cellular redox homeostasis. Trx proteins are widely distributed across various subcellular locations, and they play a crucial role in responding to cellular stresses. In this study, seven CaTrxh-type genes present in pepper were identified and the CaTrxh-type family was classified into three subgroups. CaTrxh genes responded to heat stress. Moreover, subcellular locations of the CaTrxh family exhibited dynamic patterns in normal conditions, and we observed relocalizations in heat stress conditions. Each CaTrxh family protein member formed homo-/heteromeric protein complexes in BiFC assay. Unexpectedly, subgroup III CaTrxh9 and CaTrxh10 can recruit subgroup I and II CaTrxh proteins into the plasma membrane. Thus, the function of the CaTrxh-type family is expected to play a protective role in the cell in response to high-temperature stress via protein complex formations. CaTrxh may have potential applications in the development of crops with enhanced tolerance to oxidative stress.

NMR-Based Metabolomic Analysis of Biotic Stress Responses in the Traditional Korean Landrace Red Pepper (Capsicum annuum var. annuum, cv. Subicho).

Korean landrace red peppers (Capsicum annuum var. Subicho), such as the traditional representative Subicho variety, are integral to Korean foods and are often consumed raw or used as a dried powder for cuisine. However, the known vulnerability of local varieties of landrace to biotic stresses can compromise their quality and yield. We employed nuclear magnetic resonance (NMR) spectroscopy coupled with a multivariate analysis to uncover and compare the metabolomic profiles of healthy and biotic-stressed Subicho peppers. We identified 42 metabolites, with significant differences between the groups. The biotic-stressed Subicho red peppers exhibited lower sucrose levels but heightened concentrations of amino acids, particularly branched-chain amino acids (valine, leucine, and isoleucine), suggesting a robust stress resistance mechanism. The biotic-stressed red peppers had increased levels of TCA cycle intermediates (acetic, citric, and succinic acids), nitrogen metabolism-related compounds (alanine, asparagine, and aspartic acid), aromatic amino acids (tyrosine, phenylalanine, and tryptophan), and γ-aminobutyric acid. These findings reveal the unique metabolic adaptations of the Subicho variety, underscoring its potential resilience to biotic stresses. This novel insight into the stress response of the traditional Subicho pepper can inform strategies for developing targeted breeding programs and enhancing the quality and economic returns in the pepper and food industries.

Efficient In Vitro Regeneration System and Comparative Transcriptome Analysis Offer Insight into the Early Development Characteristics of Explants from Cotyledon with Partial Petiole in Small-Fruited Pepper (Capsicum annuum).

In our research, we utilized six small-fruited pepper germplasms as materials, selected cotyledons with the petiole and hypocotyls as explants, and conducted in vitro regeneration studies. Our outcomes specify that the most suitable explant is cotyledon with the petiole, and the suitable genotype is HNUCA341. The optimal medium for inducing and elongating adventitious buds for this genotype is Murashige and Skoog medium (MS) + 9.12 µM Zeatin (ZT) + 0.57 µM 3-Indoleacetic acid (IAA), with a bud induction rate of 44.4%. The best rooting induction medium is MS + 1.14 µM IAA, with a rooting rate of 86.7%. Research on the addition of exogenous hormones has revealed that the induction speed of buds in small-fruited pepper (HNUCA341) in the combination of ZT and IAA hormones (abbreviated as ZI) is quicker, and the induction effect is better. The histological observations indicate that ZI treatment accelerates the initiation of explant division and differentiation, causing a shorter duration of vascular-bundle tissue production. The plant hormone signaling pathway was significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, including ARR9 (LOC107843874, LOC107843885), ARR4 (LOC107848380, LOC107862455), AHK4 (LOC107870540), AHP1 (LOC107839518), LAX2 (LOC107846008), SAUR36 (LOC107852624), IAA8 (LOC107841020), IAA16 (LOC107839415), PYL4 (LOC107843441), and PYL6 (LOC107871127); these significantly enriched genes may be associated with in vitro regeneration. In addition, the carbon metabolism pathway and plant mitogen-activated protein kinase (MAPK) signaling pathway are also significantly enriched in KEGG. The results of the Gene Ontology (GO) analysis revealed that differentially expressed genes related to carbon metabolism and fixation, photosynthesis and MAPK signaling pathways were upregulated under ZI treatment. It was found that they might be associated with enhanced regeneration in vitro. Furthermore, we also screened out differentially expressed transcription factors, primarily from the MYB, bHLH, AP2/ERF, and NAC families. Overall, our work accumulated important data for the in-depth analysis of the molecular mechanism of in vitro regeneration of pepper, and provides valuable germplasm for establishing an efficient stable pepper genetic-transformation system based on tissue culture.