Colletotrichum caladii sp. nov. Causing Anthracnose Leaf Spot of Caladium × hortulanum (Araceae) in Florida, U.S.A.

Caladium (Caladium × hortulanum) is an ornamental plant popular for its variable and colorful foliage. In 2020, plants showing leaf spots and blight, typical of anthracnose, were found in a field trial at the University of Florida's Gulf Coast Research and Education Center in Wimauma, Florida, U.S.A. Leaf samples consistently yielded a Colletotrichum-like species with curved conidia and abundant setae production in the acervuli. The internal transcribed spacer (ITS), partial sequences of the glyceraldehyde-3-phosphate dehydrogenase gene (gapdh), actin gene (act), chitin synthase 1 gene (chs-1), beta-tubulin gene (tub2), and histone3 gene (his3) were amplified and sequenced. BLASTN searches in the NCBI GenBank database revealed similarities to species of the Colletotrichum truncatum species complex. Phylogenetic analyses using multilocus sequence data supports a distinct species within this complex, with the closest related species being C. curcumae. Based on morphological and phylogenetic analyses, a new species of Colletotrichum, named C. caladii, is reported. Pathogenicity assays and subsequent isolation confirmed that this species was the causal agent of the disease.

Pseudocercospora pancratii Causing Leaf Spots on Commercial Blackberry (Rubus sp.) in Florida.

Blackberry (Rubus L. subgenus Rubus Watson) is a deciduous berry crop that is the fourth most economically important berry crop, and its production is expanding in the southeastern United States. However, since most commercially available cultivars were bred under temperate conditions, they are not always well adapted and could be threatened by new pathogen populations inhabiting subtropical areas. In 2017, plants showing purple or brown leaf spots and angular-to-irregular lesions on both leaf surfaces, with clusters of black conidiophores at the center, were observed in a field trial at the University of Florida's Gulf Coast Research and Education Center (UF/GCREC) in Wimauma, FL. A fungus resembling Cercospora/Pseudocercospora was isolated from the lesions. The ribosomal DNA internal transcribed spacers, the translation elongation factor 1-alpha, and the actin genes were amplified and sequenced. Based on the phylogenetic analysis, the closest related species was Pseudocercospora pancratii. Pathogenicity assays and subsequent reisolation confirmed that this species is the causal agent of the disease. Among eight cultivars screened, no complete resistance was found. However, 'Osage' was the least susceptible, and 'Kiowa' was the most susceptible. This study is the first report of P. pancratii causing leaf spots on blackberry worldwide, and it may help shape future research into disease epidemiology and management for a crop that is rapidly expanding but has very limited disease information currently available for Florida growers.

First Report of Leaf Rust on Blackberry (Rubus spp.) Caused by Kuehneola uredinis in Florida.

During the fall of 2020 and summer of 2021, symptoms of leaf rust were observed on blackberry plants of 'Kiowa', and breeding line 1734 (progeny of 'Natchez' and Arapaho') in a field trial at the University of Florida, Wimauma, FL. Symptoms consisted of small chlorotic spots (1 to 3 mm) on the upper side of the leaf, while the underside had yellow-orange pustules. Disease incidence was up to 100% on both 'Kiowa' and the breeding line 1734, and severity was up to 20% with most of the symptoms observed on older leaves. Two isolates were collected from 'Kiowa' and one from the breeding line 1734 for further investigation. Isolates were maintained and multiplied on healthy 'Kiowa' plants in growth chambers (25 ºC and 12-12 h photoperiod). Uredinia (n=30) were erumpent and ranged from 90 to 320 µm (Average=285 µm, SD=5.3 µm) in diameter. Urediniospores (n=50) were obovoid, yellow, and ranged from 24 to 36 µm long (Average=32 µm, SD=3.2 µm) and 22 to 30 µm wide (Average=28 µm, SD=2.5 µm). Based on morphology and literature, the pathogen was tentatively identified as Kuehneola uredinis (Link) Arth (Arthur 1906; Shands et al., 2018). Spores from a single uredinium of each isolate were collected with a needle and suspended in 50 µL of molecular biology-grade water yielding a final concentration of approximately 5 x 104 spores/mL. Two µL of each spore suspension was used for the PCR reactions. Two DNA fragments were amplified using the primers Rust2inv and LR6, and Rust18S-R and NS1 for the 5.8S-ITS2-28S gene region of rDNA (1,755 bp) and partial 18S gene region of rDNA (2,684 bp), respectively. The amplified products of the partial 28S gene region were sequenced with the primers LR3 and LR0R, and the 18S gene region with NS5, NS6, and NS4 (Aime 2006). DNA sequences were deposited in GenBank (accession nos. OK509845 - OK509848). BLASTn searches revealed that the isolates were 100% identical to K. uredinis reported causing leaf rust on blackberry in California (1044/1044bp, and 1540/1540bp for accession numbers MF158087, and MF158088, respectively). To test for pathogenicity, blackberry cultivars Kiowa, Natchez, Osage, Ouachita, Ponca, Prime-Ark® 45, Prime-Ark® Freedom, Prime-Ark® Traveler, and Prime-Ark® Horizon were inoculated. Five plants of each cultivar were inoculated with a mixture of spores of the three isolates, and two plants of each cultivar were used as controls. Spores were washed from leaves of 'Kiowa' exhibiting sporulation using a suspension of 1% Tween 20 in deionized water. The final concentration of the inoculum was adjusted to 104 spores/mL. Plants were inoculated in the greenhouse with a spray bottle until run-off and kept inside clear plastic boxes for 48 h. Controls were sprayed with sterile deionized water. Plants were watered by mists of 3 s every 10 min twice a week. Disease incidence and severity were evaluated weekly on five leaves per plant that had been tagged before inoculation. The experiment was repeated once. Symptoms identical to the original were only observed in 'Kiowa' and 'Prime-Ark® Freedom'. One week after inoculation, disease incidence was already 100% in both cultivars, with at least one pustule on all the inoculated leaves, and six weeks later disease severity was up to 50% (Average= 35%, SD=2.4%). To our knowledge, this is the first report of K. uredinis causing leaf rust on blackberry in Florida. This disease was reported on Rubus spp. in several U.S. states, and recently in California on Rubus ursinus (Farr and Rossman 2021; Shands et al. 2018). Blackberry is an emerging crop in Florida and efforts should be implemented to monitor the occurrence and spread of leaf rust considering that urediniospores disperse long distances by wind, especially if growers choose the susceptible cultivars 'Kiowa' and 'Prime-Ark Freedom'. The apparent resistance observed in other commercial cultivars such as 'Osage', 'Ouachita', and 'Ponca' may serve as valuable breeding parents for developing new blackberry cultivars with resistance to leaf rust.

A chromosome-scale reference genome of trifoliate orange (Poncirus trifoliata) provides insights into disease resistance, cold tolerance and genome evolution in Citrus.

Trifoliate orange (Poncirus trifoliata), a deciduous close relative of evergreen Citrus, has important traits for citrus production, including tolerance/resistance to citrus greening disease (Huanglongbing, HLB) and other major diseases, and cold tolerance. It has been one of the most important rootstocks, and one of the most valuable sources of resistance and tolerance genes for citrus. Here we present a high-quality, chromosome-scale genome assembly of P. trifoliata. The 264.9-Mb assembly contains nine chromosomal pseudomolecules with 25 538 protein-coding genes, covering 97.2% of the estimated gene space. Comparative analyses of P. trifoliata and nine Citrus genomes revealed 605 species-specific genes and six rapidly evolving gene families in the P. trifoliata genome. Poncirus trifoliata has evolved specific adaptation in the C-repeat/DREB binding factor (CBF)-dependent and CBF-independent cold signaling pathways to tolerate cold. We identified candidate genes within quantitative trait loci for HLB tolerance, and at the loci for resistance to citrus tristeza virus and citrus nematode. Genetic diversity analysis of Poncirus accessions and Poncirus/Citrus hybrids shows a narrow genetic base in the US germplasm collection, and points to the importance of collecting and preserving more natural genetic variation. Two phenotypically divergent Poncirus accessions are found to be clonally related, supporting a previous conjecture that dwarf Flying Dragon originated as a mutant of a non-dwarfing type. The high-quality genome reveals features and evolutionary insights of Poncirus, and it will serve as a valuable resource for genetic, genomic and molecular research and manipulation in citrus.

Sequencing and analysis of gerbera daisy leaf transcriptomes reveal disease resistance and susceptibility genes differentially expressed and associated with powdery mildew resistance.

BACKGROUND: RNA sequencing has been widely used to profile genome-wide gene expression and identify candidate genes controlling disease resistance and other important traits in plants. Gerbera daisy is one of the most important flowers in the global floricultural trade, and powdery mildew (PM) is the most important disease of gerbera. Genetic improvement of gerbera PM resistance has become a crucial goal in gerbera breeding. A better understanding of the genetic control of gerbera resistance to PM can expedite the development of PM-resistant cultivars. RESULTS: The objectives of this study were to identify gerbera genotypes with contrasting phenotypes in PM resistance and sequence and analyze their leaf transcriptomes to identify disease resistance and susceptibility genes differentially expressed and associated with PM resistance. An additional objective was to identify SNPs and SSRs for use in future genetic studies. We identified two gerbera genotypes, UFGE 4033 and 06-245-03, that were resistant and susceptible to PM, respectively. De novo assembly of their leaf transcriptomes using four complementary pipelines resulted in 145,348 transcripts with a N50 of 1124 bp, of which 67,312 transcripts contained open reading frames and 48,268 were expressed in both genotypes. A total of 494 transcripts were likely involved in disease resistance, and 17 and 24 transcripts were up- and down-regulated, respectively, in UFGE 4033 compared to 06-245-03. These gerbera disease resistance transcripts were most similar to the NBS-LRR class of plant resistance genes conferring resistance to various pathogens in plants. Four disease susceptibility transcripts (MLO-like) were expressed only or highly expressed in 06-245-03, offering excellent candidate targets for gene editing for PM resistance in gerbera. A total of 449,897 SNPs and 19,393 SSRs were revealed in the gerbera transcriptomes, which can be a valuable resource for developing new molecular markers. CONCLUSION: This study represents the first transcriptomic analysis of gerbera PM resistance, a highly important yet complex trait in a globally important floral crop. The differentially expressed disease resistance and susceptibility transcripts identified provide excellent targets for development of molecular markers and genetic maps, cloning of disease resistance genes, or targeted mutagenesis of disease susceptibility genes for PM resistance in gerbera.

Parts-Prospecting for a High-Efficiency Thiamin Thiazole Biosynthesis Pathway.

Plants synthesize the thiazole precursor of thiamin (cThz-P) via THIAMIN4 (THI4), a suicide enzyme that mediates one reaction cycle and must then be degraded and resynthesized. It has been estimated that this THI4 turnover consumes 2% to 12% of the maintenance energy budget and that installing an energy-efficient alternative pathway could substantially increase crop yield potential. Available data point to two natural alternatives to the suicidal THI4 pathway: (i) nonsuicidal prokaryotic THI4s that lack the active-site Cys residue on which suicide activity depends, and (ii) an uncharacterized thiazole synthesis pathway in flowers of the tropical arum lily Caladium bicolor that enables production and emission of large amounts of the cThz-P analog 4-methyl-5-vinylthiazole (MVT). We used functional complementation of an Escherichia coli ΔthiG strain to identify a nonsuicidal bacterial THI4 (from Thermovibrio ammonificans) that can function in conditions like those in plant cells. We explored whether C. bicolor synthesizes MVT de novo via a novel route, via a suicidal or a nonsuicidal THI4, or by catabolizing thiamin. Analysis of developmental changes in MVT emission, extractable MVT, thiamin level, and THI4 expression indicated that C. bicolor flowers make MVT de novo via a massively expressed THI4 and that thiamin is not involved. Functional complementation tests indicated that C. bicolor THI4, which has the active-site Cys needed to operate suicidally, may be capable of suicidal and - in hypoxic conditions - nonsuicidal operation. T. ammonificans and C. bicolor THI4s are thus candidate parts for rational redesign or directed evolution of efficient, nonsuicidal THI4s for use in crop improvement.

Plant-parasitic nematodes associated with the root zone of hop cultivars planted in a Florida field soil.

In early 2016, hop plants were introduced into Florida. By late 2016, the hop plants were showing stunted growth and were heavily parasitized by Meloidogyne javanica. In this study, we determined host susceptibility of 14 hop cultivars to M. javanica in a greenhouse experiment and monitored population development of plant-parasitic nematode species in the root zone of 17 hop cultivars planted in three newly established hop yards in Florida. Plant-parasitic nematodes in the rooting zone soil of field grown hop plants included M. javanica, Pratylenchus brachyurus, Paratrichodorus minor, Belonolaimus longicaudatus, Xiphinema setariae/vulgare complex, Mesocriconema xenoplax, and Helicotylenchus dihystera; however, soil population densities of P. minor, B. longicaudatus, X. setariae/vulgare complex, M. xenoplax, and H. dihystera remained low through the study. Root galling, M. javanica egg production, and soil population densities of M. javanica were consistently large on the 'Canadian Red Vine', 'Centennial', 'Chinook', and 'Comet' cultivars, and small on the 'Galena' and 'Triple Perle' cultivars. No differences were observed in soil population densities of P. brachyurus among hop cultivars. Overall, our study provides the first report of plant-parasitic nematode population development in the root zone on hop cultivars planted in Florida.In early 2016, hop plants were introduced into Florida. By late 2016, the hop plants were showing stunted growth and were heavily parasitized by Meloidogyne javanica. In this study, we determined host susceptibility of 14 hop cultivars to M. javanica in a greenhouse experiment and monitored population development of plant-parasitic nematode species in the root zone of 17 hop cultivars planted in three newly established hop yards in Florida. Plant-parasitic nematodes in the rooting zone soil of field grown hop plants included M. javanica, Pratylenchus brachyurus, Paratrichodorus minor, Belonolaimus longicaudatus, Xiphinema setariae/vulgare complex, Mesocriconema xenoplax, and Helicotylenchus dihystera; however, soil population densities of P. minor, B. longicaudatus, X. setariae/vulgare complex, M. xenoplax, and H. dihystera remained low through the study. Root galling, M. javanica egg production, and soil population densities of M. javanica were consistently large on the 'Canadian Red Vine', 'Centennial', 'Chinook', and 'Comet' cultivars, and small on the 'Galena' and 'Triple Perle' cultivars. No differences were observed in soil population densities of P. brachyurus among hop cultivars. Overall, our study provides the first report of plant-parasitic nematode population development in the root zone on hop cultivars planted in Florida.

Characterization of Colletotrichum Species Causing Anthracnose of Pomegranate in the Southeastern United States.

Anthracnose fruit rot and leaf blight caused by Colletotrichum species are important diseases of pomegranate in the southeastern United States. In this study, 26 isolates from pomegranate were identified based on pathological and molecular characterization. Isolates were identified to species based on multilocus sequence analysis with the internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase, ß-tubulin, and chitin synthase genomic genes. Pomegranate isolates grouped within the C. acutatum and C. gloeosporioides species complexes, with more than 73% belonging to the latter group. Three species were identified within the C. acutatum species complex (C. nymphaeae [n = 5], C. fioriniae [n = 1], and C. simmondsii [n = 1]), and three other species were identified within the C. gloeosporioides species complex (C. theobromicola [n = 11], C. siamense [n = 6], and C. gloeosporioides [n = 2]). Inoculations of pomegranate fruit showed that isolates from the C. acutatum species complex were more aggressive than isolates from the C. gloeosporioides species complex. Interestingly, opposite results were observed when leaves of rooted pomegranate cuttings were inoculated. In addition, Colletotrichum isolates from pomegranate, strawberry, blueberry, mango, and citrus were cross-pathogenic when inoculated to fruit. This is the first study identifying six different species of Colletotrichum causing pomegranate leaf blight and fruit anthracnose in the southeastern United States and the potential cross-pathogenic capability of pomegranate isolates to other commercially important crops.

Comparative Analysis of Impatiens Leaf Transcriptomes Reveal Candidate Genes for Resistance to Downy Mildew Caused by Plasmopara obducens.

Impatiens downy mildew (IDM) is a devastating disease to garden impatiens. A good understanding of IDM resistance in New Guinea impatiens is essential for improving garden impatiens resistance to this disease. The present study was conducted to sequence, assemble, annotate and compare the leaf transcriptomes of two impatiens cultivars differing in resistance to IDM, reveal sequence polymorphisms and identify candidate genes for IDM resistance. RNA-Seq was performed on cultivars Super Elfin® XP Pink (SEP) and SunPatiens® Compact Royal Magenta (SPR). De novo assembly of obtained sequence reads resulted in 121,497 unigenes with an average length of 1156 nucleotides and N50 length of 1778 nucleotides. Searching the non-redundant protein and non-redundant nucleotide, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes and Clusters of Orthologous Groups and Gene Ontology databases, resulted in annotation of 57.7% to 73.6% of the unigenes. Fifteen unigenes were highly similar to disease resistance genes and more abundant in the IDM-resistant cultivar than in the susceptible cultivar. A total of 22,484 simple sequence repeats (SSRs) and 245,936 and 120,073 single nucleotide polymorphisms (SNPs) were identified from SPR and SEP respectively. The assembled transcripts and unigenes, identified disease resistance genes and SSRs and SNPs sites will be a valuable resource for improving impatiens and its IDM resistance.

De Novo Assembly, Annotation, and Characterization of Root Transcriptomes of Three Caladium Cultivars with a Focus on Necrotrophic Pathogen Resistance/Defense-Related Genes.

Roots are vital to plant survival and crop yield, yet few efforts have been made to characterize the expressed genes in the roots of non-model plants (root transcriptomes). This study was conducted to sequence, assemble, annotate, and characterize the root transcriptomes of three caladium cultivars (Caladium × hortulanum) using RNA-Seq. The caladium cultivars used in this study have different levels of resistance to Pythiummyriotylum, the most damaging necrotrophic pathogen to caladium roots. Forty-six to 61 million clean reads were obtained for each caladium root transcriptome. De novo assembly of the reads resulted in approximately 130,000 unigenes. Based on bioinformatic analysis, 71,825 (52.3%) caladium unigenes were annotated for putative functions, 48,417 (67.4%) and 31,417 (72.7%) were assigned to Gene Ontology (GO) and Clusters of Orthologous Groups (COG), respectively, and 46,406 (64.6%) unigenes were assigned to 128 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. A total of 4518 distinct unigenes were observed only in Pythium-resistant "Candidum" roots, of which 98 seemed to be involved in disease resistance and defense responses. In addition, 28,837 simple sequence repeat sites and 44,628 single nucleotide polymorphism sites were identified among the three caladium cultivars. These root transcriptome data will be valuable for further genetic improvement of caladium and related aroids.

Comprehensive meta-analysis, co-expression, and miRNA nested network analysis identifies gene candidates in citrus against Huanglongbing disease.

BACKGROUND: Huanglongbing (HLB), the most devastating disease of citrus, is associated with infection by Candidatus Liberibacter asiaticus (CaLas) and is vectored by the Asian citrus psyllid (ACP). Recently, the molecular basis of citrus-HLB interactions has been examined using transcriptome analyses, and these analyses have identified many probe sets and pathways modulated by CaLas infection among different citrus cultivars. However, lack of consistency among reported findings indicates that an integrative approach is needed. This study was designed to identify the candidate probe sets in citrus-HLB interactions using meta-analysis and gene co-expression network modelling. RESULTS: Twenty-two publically available transcriptome studies on citrus-HLB interactions, comprising 18 susceptible (S) datasets and four resistant (R) datasets, were investigated using Limma and RankProd methods of meta-analysis. A combined list of 7,412 differentially expressed probe sets was generated using a Teradata in-house Structured Query Language (SQL) script. We identified the 65 most common probe sets modulated in HLB disease among different tissues from the S and R datasets. Gene ontology analysis of these probe sets suggested that carbohydrate metabolism, nutrient transport, and biotic stress were the core pathways that were modulated in citrus by CaLas infection and HLB development. We also identified R-specific probe sets, which encoded leucine-rich repeat proteins, chitinase, constitutive disease resistance (CDR), miraculins, and lectins. Weighted gene co-expression network analysis (WGCNA) was conducted on 3,499 probe sets, and 21 modules with major hub probe sets were identified. Further, a miRNA nested network was created to examine gene regulation of the 3,499 target probe sets. Results suggest that csi-miR167 and csi-miR396 could affect ion transporters and defence response pathways, respectively. CONCLUSION: Most of the potential candidate hub probe sets were co-expressed with gibberellin pathway (GA)-related probe sets, implying the role of GA signalling in HLB resistance. Our findings contribute to the integration of existing citrus-HLB transcriptome data that will help to elucidate the holistic picture of the citrus-HLB interaction. The citrus probe sets identified in this analysis signify a robust set of HLB-responsive candidates that are useful for further validation.

Assessment of Six Blackberry Cultivars Using a Combination of Metabolomics, Biological Activity, and Network Pharmacology Approaches.

Blackberries have gained considerable attention due to their high antioxidant content and potential health benefits. This study compared the metabolite profiles of six blackberry cultivars and investigated their biological activities. The metabolites extracted from blackberries were analyzed using metabolomics, and their biological activities and mechanisms were confirmed using in vitro models and network pharmacology. Among the cultivars examined, "Kiowa" ripe berries exhibited the highest antioxidant and anti-inflammatory activities. These effects were primarily attributed to the accumulation of flavonoids (quercitrin and luteolin) and anthocyanin (cyanidin 3-O-glucoside) in the phenylpropanoid pathway. Furthermore, our research identified 13 blackberry metabolites that interacted with 31 genes, including AKT1, CASP3, JUN, MAPK8, NOS3, NQO1, and HMOX1 which play roles in reducing oxidative stress, protecting cells from damage, and suppressing inflammation. These findings suggest that blackberry metabolites, such as quercitrin, luteolin, and cyanidin 3-O-glucoside, may exert therapeutic effects by modulating specific genes and pathways associated with antioxidant and anti-inflammatory responses. This research is promising not only for plant breeders but also for those interested in harnessing the health-promoting properties of blackberries.

Landscape and female fertility evaluation of seven heavenly bamboo cultivars as potential non-invasive alternatives to the wildtype.

In recent years, breeding initiatives have been made to reduce the fecundity of invasive plants leading to sterile cultivars. The wildtype form of heavenly bamboo (Nandina domestica Thunb. (Berberidaceae) and seven cultivars were evaluated for landscape performance, fruit production and seed viability at three sites in Florida located in southwest, northcentral, and north Florida. For heavenly bamboo cultivars in north Florida, 'Emerald Sea', 'Greray' (Sunray®), 'Lemon-Lime', 'Murasaki' (Flirt™), 'SEIKA' (Obsession™), and 'Twilight' performed well throughout much of the study with average visual quality ratings between 3.54 and 4.60 (scale of 1 to 5). In northcentral Florida heavenly bamboo cultivars are 'Emerald Sea', 'Greray', 'Lemon-Lime', 'Murasaki', 'SEIKA', and 'Twilight' performed well throughout much of the study with average quality ratings between 4.49 and 4.94. In southwest Florida, 'Emerald Sea', 'Greray', 'Lemon-Lime', 'Murasaki', and 'SEIKA' performed well with average quality ratings between 3.40 and 4.83. At all three sites, 'Emerald Sea' and the wildtype were similar in size, having the greatest growth indices compared to medium-sized cultivars ('Greray', 'Lemon-Lime', 'Twilight' and 'SEIKA') and dwarf-sized cultivars ('Chime' and 'Murasaki'). For three consecutive fall-winter seasons of the study, 'Chime', 'Greray', and 'Lemon-Lime' heavenly bamboo did not fruit at any of the study sites. Among the three sites, 'Murasaki' had 97.7% to 99.9% fruit reduction, 'SEIKA' had 97.7% to 100% fruit reduction, and 'Twilight' had 95.9% to 100% fruit reduction compared to the wildtype at respective sites. Seeds collected from low fruiting cultivars ('Murasaki', 'SEIKA', and 'Twilight') had 33.3% to 66.7% viability, as determined by tetrazolium tests. In comparison, 'Emerald Sea' produced as much, if not more, fruit as the wildtype, especially in northern Florida, with seed viability ranging from 6.7% to 29.0% among sites. Nuclear DNA content of cultivars were comparable to the wildtype, suggesting they are diploids. These findings identified four low to no fruiting heavenly bamboo cultivars recommended for landscape use ('Lemon-Lime', 'SEIKA', 'Murasaki', and 'Greray').

Engineered dsRNA-protein nanoparticles for effective systemic gene silencing in plants.

Long-distance transport or systemic silencing effects of exogenous biologically active RNA molecules in higher plants have not been reported. Here, we report that cationized bovine serum albumin (cBSA) avidly binds double-stranded beta-glucuronidase RNA (dsGUS RNA) to form nucleic acid-protein nanocomplexes. In our experiments with tobacco and poplar plants, we have successfully demonstrated systemic gene silencing effects of cBSA/dsGUS RNA nanocomplexes when we locally applied the nanocomplexes from the basal ends of leaf petioles or shoots. We have further demonstrated that the cBSA/dsGUS RNA nanocomplexes are highly effective in silencing both the conditionally inducible DR5-GUS gene and the constitutively active 35S-GUS gene in leaf, shoot, and shoot meristem tissues. This cBSA/dsRNA delivery technology may provide a convenient, fast, and inexpensive tool for characterizing gene functions in plants and potentially for in planta gene editing.

Transcriptome analysis of Lantana camara flower petals reveals candidate anthocyanin biosynthesis genes mediating red flower color development.

Flower color plays a crucial role in the appeal and selection of ornamental plants, directly influencing breeding strategies and the broader horticulture industry. Lantana camara, a widely favored flowering shrub, presents a rich palette of flower colors. Yet, the intricate molecular mechanisms governing this color variation in the species have remained largely unidentified. With the aim of filling this gap, this study embarked on a comprehensive de novo transcriptome assembly and differential gene expression analysis across 3 distinct lantana accessions, each showcasing a unique flower color. By harnessing the capabilities of both PacBio and Illumina sequencing platforms, a robust transcriptome assembly, encompassing 123,492 gene clusters and boasting 94.2% BUSCO completeness, was developed. The differential expression analysis unveiled 72,862 unique gene clusters that exhibited varied expression across different flower stages. A pronounced upregulation of 8 candidate core anthocyanin biosynthesis genes in the red-flowered accession was uncovered. This was further complemented by an upregulation of candidate MYB75 (PAP1) and bHLH42 (TT8) transcription factors. A candidate carotenoid cleavage dioxygenase (CCD4a) gene cluster also manifested a marked upregulation in white flowers. The study unveils the molecular groundwork of lantana's flower color variation, offering insights for future research and potential applications in breeding ornamental plants with desired color traits.

A chromosome-level phased genome enabling allele-level studies in sweet orange: a case study on citrus Huanglongbing tolerance.

Sweet orange originated from the introgressive hybridizations of pummelo and mandarin resulting in a highly heterozygous genome. How alleles from the two species cooperate in shaping sweet orange phenotypes under distinct circumstances is unknown. Here, we assembled a chromosome-level phased diploid Valencia sweet orange (DVS) genome with over 99.999% base accuracy and 99.2% gene annotation BUSCO completeness. DVS enables allele-level studies for sweet orange and other hybrids between pummelo and mandarin. We first configured an allele-aware transcriptomic profiling pipeline and applied it to 740 sweet orange transcriptomes. On average, 32.5% of genes have a significantly biased allelic expression in the transcriptomes. Different cultivars, transgenic lineages, tissues, development stages, and disease status all impacted allelic expressions and resulted in diversified allelic expression patterns in sweet orange, but particularly citrus Huanglongbing (HLB) shifted the allelic expression of hundreds of genes in leaves and calyx abscission zones. In addition, we detected allelic structural mutations in an HLB-tolerant mutant (T19) and a more sensitive mutant (T78) through long-read sequencing. The irradiation-induced structural mutations mostly involved double-strand breaks, while most spontaneous structural mutations were transposon insertions. In the mutants, most genes with significant allelic expression ratio alterations (≥1.5-fold) were directly affected by those structural mutations. In T19, alleles located at a translocated segment terminal were upregulated, including CsDnaJ, CsHSP17.4B, and CsCEBPZ. Their upregulation is inferred to keep phloem protein homeostasis under the stress from HLB and enable subsequent stress responses observed in T19. DVS will advance allelic level studies in citrus.

Construction of a genome-wide genetic linkage map and identification of quantitative trait loci for powdery mildew resistance in Gerbera daisy.

Powdery mildew (PM) is a common fungal disease in many important crops. The PM caused by Podosphaera xanthii has been the most challenging problem in commercial Gerbera (Gerbera hybrida) production globally, often leading to severe losses of crop yield and quality. A small number of PM-resistant breeding lines and cultivars have been reported in Gerbera, but the underlying genetics for PM resistance in Gerbera is largely unknown. Scarcity of genomic resources such as genetic linkage maps and molecular markers has severely hindered the effort to understand the genetic basis and locate loci controlling PM resistance in Gerbera. This study aimed to construct a genome-wide genetic linkage map, identify quantitative trait loci (QTL), and molecular markers for PM resistance in Gerbera. A segregating mapping population was developed by crossing PM-resistant and -susceptible Gerbera breeding lines, genotyped by sequencing, and phenotyped for PM resistance. A genome-wide genetic linkage map constructed with 791 single polymorphic site (SNP) markers spans 1912.30 cM across 27 linkage groups (LG) and reaches a density of 1 marker per 2.42 cM. One major consistent QTL was discovered in LG16, explaining more than 16.6% of the phenotypic variance for PM resistance. The QTL was tagged with two flanking SNP markers. The availability of this genetic linkage map will be very useful for locating and tagging QTLs for other important traits in Gerbera, and the newly discovered QTL and SNP markers will enable development of molecular markers for improving Gerbera for resistance to PM.

Selection and Evaluation of a Thornless and HLB-Tolerant Bud-Sport of Pummelo Citrus With an Emphasis on Molecular Mechanisms.

The selection of elite bud-sports is an important breeding approach in horticulture. We discovered and evaluated a thornless pummelo bud-sport (TL) that grew more vigorously and was more tolerant to Huanglongbing (HLB) than the thorny wild type (W). To reveal the underlying molecular mechanisms, we carried out whole-genome sequencing of W, and transcriptome comparisons of W, TL, and partially recovered thorny "mutants" (T). The results showed W, TL, and T varied in gene expression, allelic expression, and alternative splicing. Most genes/pathways with significantly altered expression in TL compared to W remained similarly altered in T. Pathway and gene ontology enrichment analysis revealed that the expression of multiple pathways, including photosynthesis and cell wall biosynthesis, was altered among the three genotypes. Remarkably, two polar auxin transporter genes, PIN7 and LAX3, were expressed at a significantly lower level in TL than in both W and T, implying alternation of polar auxin transport in TL may be responsible for the vigorous growth and thornless phenotype. Furthermore, 131 and 68 plant defense-related genes were significantly upregulated and downregulated, respectively, in TL and T compared with W. These genes may be involved in enhanced salicylic acid (SA) dependent defense and repression of defense inducing callose deposition and programmed cell death. Overall, these results indicated that the phenotype changes of the TL bud-sport were associated with tremendous transcriptome alterations, providing new clues and targets for breeding and gene editing for citrus improvement.

Integration of early disease-resistance phenotyping, histological characterization, and transcriptome sequencing reveals insights into downy mildew resistance in impatiens.

Downy mildew (DM), caused by obligate parasitic oomycetes, is a destructive disease for a wide range of crops worldwide. Recent outbreaks of impatiens downy mildew (IDM) in many countries have caused huge economic losses. A system to reveal plant-pathogen interactions in the early stage of infection and quickly assess resistance/susceptibility of plants to DM is desired. In this study, we established an early and rapid system to achieve these goals using impatiens as a model. Thirty-two cultivars of Impatiens walleriana and I. hawkeri were evaluated for their responses to IDM at cotyledon, first/second pair of true leaf, and mature plant stages. All I. walleriana cultivars were highly susceptible to IDM. While all I. hawkeri cultivars were resistant to IDM starting at the first true leaf stage, many (14/16) were susceptible to IDM at the cotyledon stage. Two cultivars showed resistance even at the cotyledon stage. Histological characterization showed that the resistance mechanism of the I. hawkeri cultivars resembles that in grapevine and type II resistance in sunflower. By integrating full-length transcriptome sequencing (Iso-Seq) and RNA-Seq, we constructed the first reference transcriptome for Impatiens comprised of 48,758 sequences with an N50 length of 2060 bp. Comparative transcriptome and qRT-PCR analyses revealed strong candidate genes for IDM resistance, including three resistance genes orthologous to the sunflower gene RGC203, a potential candidate associated with DM resistance. Our approach of integrating early disease-resistance phenotyping, histological characterization, and transcriptome analysis lay a solid foundation to improve DM resistance in impatiens and may provide a model for other crops.

Heterologous Expression of the Constitutive Disease Resistance 2 and 8 Genes from Poncirus trifoliata Restored the Hypersensitive Response and Resistance of Arabidopsis cdr1 Mutant to Bacterial Pathogen Pseudomonas syringae.

Huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus worldwide. In the United States, this disease is associated with a phloem-restricted bacterium, Candidatus Liberibacter asiaticus. Commercial citrus cultivars are susceptible to HLB, but Poncirus trifoliata, a close relative of Citrus, is highly tolerant of HLB. Isolating P. trifoliata gene(s) controlling its HLB tolerance followed by expressing the gene(s) in citrus is considered a potential cisgenic approach to engineering citrus for tolerance to HLB. Previous gene expression studies indicated that the constitutive disease resistance (CDR) genes in P. trifoliata (PtCDRs) may play a vital role in its HLB tolerance. This study was designed to use Arabidopsis mutants as a model system to confirm the function of PtCDRs in plant disease resistance. PtCDR2 and PtCDR8 were amplified from P. trifoliata cDNA and transferred into the Arabidopsis cdr1 mutant, whose resident CDR1 gene was disrupted by T-DNA insertion. The PtCDR2 and PtCDR8 transgenic Arabidopsis cdr1 mutant restored its hypersensitive response to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) expressing avrRpt2. The defense marker gene PATHOGENESIS RELATED 1 (PR1) expressed at much higher levels in the PtCDR2 or PtCDR8 transgenic cdr1 mutant than in the non-transgenic cdr1 mutant with or without pathogen infection. Multiplication of Pst DC3000 bacteria in Arabidopsis was inhibited by the expression of PtCDR2 and PtCDR8. Our results showed that PtCDR2 and PtCDR8 were functional in Arabidopsis and played a positive role in disease resistance and demonstrated that Arabidopsis mutants can be a useful alternate system for screening Poncirus genes before making the time-consuming effort to transfer them into citrus, a perennial woody plant that is highly recalcitrant for Agrobacterium or biolistic-mediated transformation.