Towards realizing nano-enabled precision delivery in plants.

Nanocarriers (NCs) that can precisely deliver active agents, nutrients and genetic materials into plants will make crop agriculture more resilient to climate change and sustainable. As a research field, nano-agriculture is still developing, with significant scientific and societal barriers to overcome. In this Review, we argue that lessons can be learned from mammalian nanomedicine. In particular, it may be possible to enhance efficiency and efficacy by improving our understanding of how NC properties affect their interactions with plant surfaces and biomolecules, and their ability to carry and deliver cargo to specific locations. New tools are required to rapidly assess NC-plant interactions and to explore and verify the range of viable targeting approaches in plants. Elucidating these interactions can lead to the creation of computer-generated in silico models (digital twins) to predict the impact of different NC and plant properties, biological responses, and environmental conditions on the efficiency and efficacy of nanotechnology approaches. Finally, we highlight the need for nano-agriculture researchers and social scientists to converge in order to develop sustainable, safe and socially acceptable NCs.

Innovative stain-free technique for high-resolution imaging of virus particles via standard transmission electron microscopy.

This research presents a groundbreaking approach in virus-related research, addressing challenges in electron microscopy (EM). This imaging technique has been crucial in exploring virus structures; however, traditional methods involve complex sample preparations and the risk of contamination. Herein, we introduce an approach that overcomes these obstacles, enabling high-resolution virus imaging without toxic staining procedures. Focusing on Begomovirus particles, an economically significant plant virus genus, our images confirm their non-enveloped structure and their twin icosahedral symmetry. Our methods involve sample collection, purification, and crystallization, followed by transmission electron microscopy - selected area electron diffraction (TEM-SAED) analysis. Notably, this study achieves 2D and 3D virus imaging through standard TEM, providing a new avenue for virus structure analysis and advancing virus-related research. Remarkable high image quality stemmed from the crystallization process, offering exciting possibilities for improving virus research and diagnosis while eliminating staining limitations.

Tiny but Mighty: Nanoscale Materials in Plant Disease Management.

Nanoscale materials are promising tools for managing plant diseases and are becoming important players in the current agritech revolution. However, adopting modern methodologies requires a broad understanding of their effectiveness in solving target problems and their effects on the environment and food chain. Furthermore, it is paramount that such technologies are mechanistically and economically feasible for growers to adopt in order to be sustainable in the long run. This Feature Article summarizes the latest findings on the role of nanoscale materials in managing agricultural plant pathogens. Herein, we discussed the benefits and limitations of using nanoscale materials in plant disease management and their potential impacts on the environment and global food security.

Viability and oxidative stress of dental pulp cells after indirect application of chemomechanical agents: An in vitro study.

AIM: This study evaluated the transdentinal cytotoxic effects of enzymatic agents (EA) for chemomechanical carious tissue removal on human dental pulp cells. METHODOLOGY: The groups were based on the performed dentine treatments (n = 8): G1: Positive Control (PC - no treatment); G2: Negative Control (NC - 35% H2 O2 for 2 min); G3: Brix 3000™ (BX) for 30 s; G4: BX for 2 min; G5: Papacarie Duo™ (PD) for 30 s; G6: PD for 2 min. The cells were evaluated for viability (VB; MTT assay) and production of reactive oxygen species (ROS; DCFH-DA assay) and nitric oxide (NO; Griess reagent). A scanning electron microscope provided morphological chemical analyses and energy-dispersive X-ray spectroscopy. The data were submitted to the one-way anova statistical test complemented by Tukey (p < .05). RESULTS: Cell viability decreased by 21.1% and 58.4% in G5 and G6, respectively. ROS production in G3 and G4 maintained basal levels but increased by 171.2% and 75.1% in G5 and G6, respectively. CONCLUSIONS: The Brix3000™ enzymatic agent did not cause indirect cytotoxic effects on pulp cells, regardless of the application time. Conversely, Papacarie Duo™ reduced viability and increased ROS production by pulp cells.

First report of Lasiodiplodia brasiliensis causing root rot in melon plants in northeastern Brazil.

Melon (Cucumis melo L.) is the second most exported fruit in Brazil with an annual production of 27.5 million tons (FAO 2023). From September 2019 through February 2020, 50-day-old melon plants started showing root rot symptoms (dark-brow necrotic zones in their roots that extended to the collar zone) in northeastern Brazil, 30% of the plants in the fields were affected by the disease. The fields are in clay soil where melon, in monoculture, is produced all year long with three cycles of the culture per year. A total of 132 samples from "Yellow" and "Cantaloupe" cultivars were collected from four melon fields (4°59'45.3"S, 37°33'39.7"W; 4°57'10.2"S, 37°31'37.1"W; 5°38'17.9"S, 37°56'27.7"W; and 5°00'25.5"S, 37°23'55.3"W). Small pieces of diseased tissues were surface disinfested in 70% ethanol for 30 sec, in 2% sodium hypochlorite for 1 min, washed in sterilized distilled water, plated on a PDA Petri dishes with tetracycline (0.05g/L), and incubated for seven days at 28 ± 2 ºC. Nine representative isolates were selected for downstream analysis. Colonies were white and later became dark gray, pycnidia and conidia were produced after 30 days ofncubation at 25°C under near-UV light in water-agar medium. Conidia were hyaline when immature and dark brown when mature, ranging from cylindrical subovoid to ellipsoidal and septate to non-septate, and with an average size of 12.54 to 21.97 µm. The colonies were morphologically identified as Lasiodiplodia sp. (Phillips et al. 2013). Total DNA from the isolates was extracted and the ITS, TUB, and TEF-1α genes (Jayawardena et al. 2019) were partially amplified by PCR, Sanger sequenced, and deposited in Genbank: ITS (OM102511 to OM102520), TUB (OR062087 to OR062094 and OR062095), and TEF-1α (OP536826 to OP536835). Blastn analysis of the partial sequences ITS (519bp), TUB (388bp), and TEF-1α (315bp) showed 100% nucleotide similarity of the isolates with sequences of L. brasiliensis and L. theobromae from the GenBank. A phylogenetic tree was constructed using the Maximum Parsimony Analysis method. All nine isolates were grouped into the L. brasiliensis clade with 71% bootstrap support, confirming the isolates's identity. Pathogenicity assays were conducted in a greenhouse using the wooden toothpick inoculation method (Nogueira et al. 2019). "Goldex" Yellow melon seedlings were used in a completely randomized experimental design, with 10 treatments (9 isolates + Mock) and six replicates, with one plant per pot. Plants were inoculated 15 days after sowing, and disease severity was evaluated 50 days after inoculation. All nine isolates caused symptoms in the assessed melon plants. The fungus was reisolated from the lesions and looked morphologically identical to the inoculated fungus, fulfilling Koch's postulates. The pathogenicity test was repeated and yielded similar results. All samples in this study were provided by melon growers who were concerned about the high incidence of root rot disease in their plantations. More research needs to be conducted to determine the epidemiology and the extension of the economic impact caused by this pathogen to melons to develop strategies for disease control to properly assist the growers's concerns. This pathogen has been reported to cause disease in other crops in Brazil, e.g., watermelon (Alves et al. 2023) and apples (Martins et al. 2018). However, to the best of our knowledge, this is the first report of L. brasiliensis causing root rot in melons in Brazil.

Biologically synthesized zinc and copper oxide nanoparticles using Cannabis sativa L. enhance soybean (Glycine max) defense against fusarium virguliforme.

In this study, zinc and copper oxide nanoparticles (NPs) were synthesized using hemp (Cannabis sativa L.) leaves (ZnONP-HL and CuONP-HL), and their antifungal potential was assessed against Fusarium virguliforme in soybean (Glycine max L.). Hemp was selected because it is known to contain large quantities of secondary metabolites that can potentially enhance the reactivity of NPs through surface property modification. Synthesizing NPs with biologically derived materials allows to avoid the use of harsh and expensive synthetic reducing and capping agents. The ZnONP-HL and CuONP-HL showed average grain/crystallite size of 13.51 nm and 7.36 nm, respectively. The biologically synthesized NPs compared well with their chemically synthesized counterparts (ZnONP chem, and CuONP chem; 18.75 nm and 10.05 nm, respectively), confirming the stabilizing role of hemp-derived biomolecules. Analysis of the hemp leaf extract and functional groups that were associated with ZnONP-HL and CuONP-HL confirmed the presence of terpenes, flavonoids, and phenolic compounds. Biosynthesized NPs were applied on soybeans as bio-nano-fungicides against F. virguliforme via foliar treatments. ZnONP-HL and CuONP-HL at 200 µg/mL significantly (p < 0.05) increased (∼ 50%) soybean growth, compared to diseased controls. The NPs improved the nutrient (e.g., K, Ca, P) content and enhanced photosynthetic indicators of the plants by 100-200%. A 300% increase in the expression of soybean pathogenesis related GmPR genes encoding antifungal and defense proteins confirmed that the biosynthesized NPs enhanced disease resistance against the fungal phytopathogen. The findings from this study provide novel evidence of systemic suppression of fungal disease by nanobiopesticides, via promoting plant defense mechanisms.

First Report of Cladosporium tenuissimum causing spot diseases on leaves and fruits of cucurbits in Brazil.

Cucurbitaceae crops are widely cultivated in the Northeast region of Brazil, which is the biggest producer of melon and watermelon in the country (Oliveira, 2020). Between November and December 2020 leaves of pumpkins (Cucurbita maxima L.) and watermelon (Citrullus lanatus L.), and leaves and fruits of melon plants (Cucumis melo L.) were collected with moderate to severe necrotic, irregular, and brown lesions from farms in the state of Rio Grande do Norte, Brazil. Fragments of diseased tissues were cut into small pieces and surface disinfested in 70% ethanol for 30 seconds, then in 2% sodium hypochlorite for 1 minute, and washed in sterile distilled water. Disinfested pieces of tissue were plated on potato dextrose agar (PDA) and incubated for seven days in the dark at 28 ± 2 °C. A total of 12 fungal isolates (four from pumpkins, one from watermelon, and seven from melons) were isolated from leaves and symptomatic fruits. All isolates in this study shared similar morphological characteristics. The colonies were dark gray to olive green in color with a velvety texture and surrounded by gray-white hyphae. The conidiophores were erect, tall, dark, and irregularly branched at the apex containing dark conidia, with 0 to 3 septa, variable in shape and size, forming chains that were often branched, globose, or subglobose with 3 to 4.5 µm in diameter. DNA from each isolate was extracted using the SDS method (Smith et al., 2001) and submitted to PCR amplification of the ITS and TEF1α regions with the primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The amplicons were sequenced and deposited in GenBank: ITS (OP493545-OP493556) and TEF1α (OP536836-OP536847). Blastn analysis of the ITS and TEF1α partial sequences revealed that all 12 isolates belong to the species Cladosporium tenuissimum, with 100% nucleotide similarity with sequences of many C. tenuissimum isolates deposited in GenBank. A phylogenetic tree was constructed using the Maximum Parsimony Analysis, with the concatenated sequences (ITS-TEF1α) on MEGAX software (version 11.0.8) (Tamura et al, 2018). All 12 isolates clustered in the same clade and were closely related to isolates A2PP5, A3I1, and XCHK2 with the respective accession numbers KU605789.1, KU605790.1, and MG873071.1 from GenBank, with 99% bootstrap support. The pathogenicity of the 12 isolates was evaluated in pumpkin and melon plants in a greenhouse. Spore suspensions (10 6 conidia/ml -1) were sprayed on the leaves of healthy seedlings until runoff, only water was sprayed on control plants as the mock, and five seedlings of each crop (melon and pumpkin) were inoculated in each treatment. All plants were covered with plastic bags for two days. Spots, similar to those observed on diseased plants in the field, developed on the inoculated leaves (after seven days from the inoculation day, no symptoms were observed on plants from the mock treatment) and the fungal morphology was identical to that observed on the originally diseased leaves, fulfilling Koch's postulate. The pathogenicity test was repeated and yielded the same results. The fact that all 12 isolates were pathogenic on pumpkin and melon leaves, indicates that many Cucurbits are susceptible to C. tenuissimum infection. Many growers in the region are reporting similar symptoms in their melon plantations and it appears that the disease incidence is getting more severe year after year, based on growers's reports. Therefore, more research needs to be conducted to determine the epidemiology and the extension of the economic impact caused by this pathogen to Cucurbits to develop strategies for disease control. To the best of our knowledge, this is the first report of C. tenuissimum causing disease in Cucurbits in Brazil.

Gathered from the Vine: A Survey of Seven Grapevine Viruses Within New England Vineyards.

Vineyards in the Southeastern New England American Viticultural Area were surveyed for the incidence of seven major viruses: grapevine leafroll-associated viruses (GLRaV-1, GLRaV-2, GLRaV-3, and GLRaV-4), grapevine fanleaf virus (GFLV), tomato ringspot virus (ToRSV), and tobacco ringspot virus (TRSV). Viruses were detected by DAS-ELISA and confirmed by RT-PCR and Sanger sequencing. Multiple viruses were present in 19 out of the 25 vineyards surveyed between 2018 and 2020. GLRaV-3 (27.59%) was the most prevalent virus followed by GLRaV-4 (14.90%), GLRaV-1 (13.52%), GLRaV-2 (11.03%), ToRSV (6.34%), GFLV (5.24%), and TRSV (2.62%). Furthermore, phylogenetic analyses of the viral partial genome sequences acquired in this study revealed that the grapevine viruses present in this area are diverse, indicating that they may have been introduced from different sources. Our findings stress the need for improving the sanitary status of planting materials to avoid the introduction and dissemination of viruses to vineyards in this important wine-producing region of New England.

Aggressivity of Different Fusarium Species Causing Fruit Rot in Melons in Brazil.

Brazil is one of the largest melon (Cucumis melo) producers in the world and most of the production is exported to international markets. Currently, over 15% of Brazilian melon shipments are lost during export transportation due to Fusarium fruit rot, which is jeopardizing the livelihood of Brazilian melon producers. We focused on understanding the aggressivity of five species of Fusarium causing fruit rot on the main types of melon produced in Brazil. We also investigated the correlation between pathogenicity and fruit quality. Experiments were performed under a completely randomized experimental design, in a 5 × 8 factorial scheme, using two methods for inoculation: deposition of discs of culture media containing fungal structures and deposition of spore suspensions in needle-punctured lesions. The fungal species used were Fusarium falciforme, F. sulawesiense, F. pernambucanum, F. kalimantanense, and Fusarium sp. Fruits of two hybrids from four types of melons, canary (Goldex and Gold Mine), piel de sapo (Grand Prix and Flecha Verde), galia (McLaren and DRG3228), and cantaloupe (SV1044MF and Bonsai), were used. Disease severity was assessed by measuring the lesions, disease severity index, fruit firmness, and degrees Brix of fruits. The five Fusarium species caused rot in the fruits of all melon hybrids studied and the aggressivity of those fungal species varied with the type and hybrid. Fruits of the hybrids McLaren and Bonsai presented the largest lesions among all melon hybrids, and hybrids of canary type (Gold Mine and Goldex) were the most tolerant to rot caused by the Fusarium species investigated. Furthermore, the greater the severity of Fusarium fruit rot, the lower the pulp firmness of the fruits, but degrees Brix did not correlate with the onset of the disease.

Evaluation of cytotoxicity and genotoxicity of a novel oxovanadium complex with orotate.

The development of new drugs based on metal complexes requires a detailed analysis of their biological endpoints. In this study, we report the genotoxic profile and influence on cell proliferation and death of the oxovanadium(IV) complex with orotic acid ([VO(C5H4N2O4)2], VO(oro)). Human hepatocellular carcinoma cells (HepG2) were the most sensitive tumor cells to VO(oro), which interfered with the integrity of cell membranes and proliferative capacity in a dose-dependent manner, inducing cell death by apoptosis. Regarding genotoxicity, VO(oro) did not induce considerable levels of DNA damage in HepG2 cells (comet test) and gene mutations (Ames test). However, it caused a statistically significant increase in the frequency of micronuclei at the highest concentration tested (12.5 µmol.L-1), indicating aneuploidy and clastogenicity. The data presented here provide information on various biological aspects of the VO(oro) complex, which may allow the elucidation of its mechanism of action as a possible therapeutic agent.

Salivary and Dental Plaque Composition in Disabled Children Who Require Home Care: A Cross-sectional Investigation.

Aims: The purpose of this study was to compare salivary and dental plaque (DP) composition between disabled children who require home care (DCHC) and a control group (CG) and to correlate it with oral and systemic health factors. Materials and Methods: This cross-sectional study included 15 DCHC and 15 healthy children (aged between 4 and 10 years). The caregivers answered a questionnaire on disease diagnosis, medical history, dental history, and oral hygiene routine. In addition to clinical examination, saliva and DP samples were collected and analyzed using attenuated total reflection-Fourier transform infrared spectroscopy. Data were collected between January and December 2019. Student's t and Kendall correlation tests were used. Results: Calculus (46.7%), bleeding on toothbrushing (53.3%), and gingival hyperplasia (40.0%) were prevalent in DCHC. The saliva of DCHC presented a higher amount of lipids and collagen and a lower amount of carbohydrates than that of the CG (P < 0.05). DP components were similar in DCHC and CG. Conclusion: DCHC presented oral comorbidities and changes in salivary composition, compared with the CG.

Cytotoxicity and dentin composition alterations promoted by different chemomechanical caries removal agents: A preliminary in vitro study.

BACKGROUND: The use of chemomechanical agents for caries removal has been indicated as a non-invasive treatment option; however, their possible deleterious effects on the dental-pulp complex have been insufficiently studied. This study assessed the direct cytotoxicity of two chemomechanical caries removal agents (Brix 3000™ - BX and Papacarie Duo™ - PD) on pulp cells from deciduous teeth, as well as to assess the morphology and chemical compositions of the dentin surface after the application of these materials. MATERIAL AND METHODS: The cells were seeded (50,000 cells/cm²) in a culture medium (DMEM with 10% fetal bovine serum - FBS). After 24 hours, the BX and PD materials were added to 1:20, 1:100, and 1:1000 dilutions. Non-exposed cells were considered as the control group. The viability test (MTT), Trypan Blue assay (TB), and cell morphology (Scanning Electron Microscopy - SEM) were performed after 24 hours of agent application. For the SEM and chemical (energy-dispersive X-ray spectrometry - EDS) dentin evaluation, 0.3-mm-thick dentin discs were obtained and divided into control group (no treatment) and surfaces covered with 37% phosphoric acid, BX, or PD. Data were compared by one-way ANOVA and Tukey's test (p<0.05). RESULTS: Decreases in cell viability and numbers of viable cells were observed for both materials, at all dilutions, when compared with the control group (p<0.05). The BX and PD materials did not cause visually perceptible changes, according to SEM, on the surfaces of dentin discs. The EDS analysis did not indicate a statistically significant difference in the levels of calcium (Ca) and phosphorus (P) between the materials and the control group (p>0.05). CONCLUSIONS: Both materials showed cytotoxicity when in direct contact with the pulp cells from deciduous teeth, and the BX material presented lower cytotoxicity than the PD material. Moreover, both materials did not significantly change the dentin composition. Key words:Cell culture, cytotoxicity, dental pulp, papacarie, primary teeth.

Nanotechnology and Plant Viruses: An Emerging Disease Management Approach for Resistant Pathogens.

Phytoviruses are highly destructive plant pathogens, causing significant agricultural losses due to their genomic diversity, rapid, and dynamic evolution, and the general inadequacy of management options. Although an increasing number of studies are being published demonstrating the efficacy of engineered nanomaterials to treat a range of plant pathogens, very little work has been done with phytoviruses. Herein, we describe the emerging field of "Nanophytovirology" as a potential management approach to combat plant viral diseases. Because of their special physiochemical properties, nanoparticles (NPs) can interact with viruses, their vectors, and the host plants in a variety of specific and useful ways. We specifically describe the potential mechanisms underlying NPs-plant-virus interactions and explore the antiviral role of NPs. We discuss the limited literature, as well as the challenges and research gaps that are instrumental to the successful development of a nanotechnology-based, multidisciplinary approach for timely detection, treatment, and prevention of viral diseases.

Memantine hydrochloride: a drug to be repurposed against Chikungunya virus?

BACKGROUND: Chikungunya fever is an endemic disease caused by the Chikungunya virus (CHIKV) to which there is no vaccine or effective antiviral drug treatment so far. Our study aimed to evaluate the potential anti-CHIKV activity of memantine hydrochloride (mtnH), a drug from the class of the aminoadamantanes approved for the treatment of Alzheimer´s disease, as a possible drug to be repurposed to the treatment of Chikungunya fever. METHODS: MtnH antiviral activity against CHIKV was determined by infecting BHK-21 cells with CHIKV-nanoluc, a virus carrying the marker nanoluciferase reporter, in the presence or absence of mtnH at concentrations ranging from 500 to 1.45 µM. The effective concentration of 50% inhibition (EC50) was calculated. Cell viability assay (determination of CC50) was also performed employing BHK-21 cells. Mutagenic assays were performed by the Salmonella Typhimurium/microsome assay (Ames test). RESULTS: MtnH presented a CC50 of 248.4 ± 31.9 µM and an EC50 of 32.4 ± 4 µM against CHIKV in vitro. The calculated selectivity index (SI) was 7.67. MtnH did not induce genetic mutation in Salmonella strains with or without an external metabolizing system. CONCLUSION: With the data herein presented, it is possible to hypothesize mtnH as a viable candidate to be repurposed as an anti-CHIKV drug. Clinical assays are, therefore, encouraged due to the promising in vitro results. The drug memantine hydrochloride is herein personified with a doubt: as a prior regulated drug against Alzheimer, could it follow the path against Chikungunya virus too?

A Fig Deal: A Global Look at Fig Mosaic Disease and its Putative Associates.

Fig mosaic disease (FMD) is a complex viral disease with which 12 viruses, including a confirmed causal agent, fig mosaic emaravirus (FMV), and three viroids are associated worldwide. FMD was first described in California in the early 1930s. Symptoms include foliar chlorosis, deformation, and mosaic patterns. FMD is disseminated by vegetative propagation, seed transmission, and vectors, including a mite, Aceria ficus. Management of the disease in fig orchards relies on scouting and elimination of infected trees. In this review, we focus on the distribution of the FMD-associated viruses and viroids by summarizing worldwide surveys and their genome structure. We also determined the full-length sequence of FMV and fig badnavirus 1 (FBV-1) isolates from Connecticut and compared the virus and viroid sequences from fig isolates. We suggest important areas of research including determining the potential synergistic effect of multiple viruses, elucidating the full-length genome sequence of each associated virus, and relating virus titer to phenotypic changes in Ficus carica.

Small RNA profiling analysis of two recombinant strains of potato virus Y in infected tobacco plants.

Plant viral infections lead to accumulation of virus-derived small interfering RNAs (vsiRNAs) as a result of host defense mechanisms. High-throughput sequencing technology enables vsiRNA profiling analyses from virus infected plants, which provide important insights into virus-host interactions. Potato virus Y (PVY) is a detrimental plant pathogen that can infect a variety of solanaceous crops, e.g., potato, tobacco, tomato, and pepper. We analyzed and characterized vsiRNAs derived from Nicotiana tabacum cv. Samsun infected with two recombinant PVY strains, N-Wi and NTN. We observed that the average percentage of vsiRNAs derived from plants infected with N-Wi was higher than from plants infected with NTN, indicating that N-Wi invokes a stronger host response than NTN in tobacco. The size distribution pattern and polarity of vsiRNAs were similar between both virus strains with the 21 and 22 nucleotide (nt) vsiRNA classes as most predominant and the sense/antisense vsiRNAs ratio nearly equal in the 20-24 nt class. However, the percentage of sense vsiRNAs was significantly higher in the 25-26 nt long vsiRNAs. Distinct vsiRNA hotspots, identifying highly abundant reads of different unique vsiRNA sequences, were observed in both viral genomes. Previous studies found an A or U bias at the 5' terminal nucleotide position of 21 nt vsiRNAs; in contrast, our analysis revealed a C and U nucleotide bias. This study provides insights that will help further elucidate differential processing of vsiRNAs in plant antiviral defense.

Transmission modes affect the population structure of potato virus Y in potato.

Transmission is a crucial part of a viral life cycle and transmission mode can have an important impact on virus biology. It was demonstrated that transmission mode can influence the virulence and evolution of a virus; however, few empirical data are available to describe the direct underlying changes in virus population structure dynamics within the host. Potato virus Y (PVY) is an RNA virus and one of the most damaging pathogens of potato. It comprises several genetically variable strains that are transmitted between plants via different transmission modes. To investigate how transmission modes affect the within-plant viral population structure, we have used a deep sequencing approach to examine the changes in the genetic structure of populations (in leaves and tubers) of three PVY strains after successive passages by horizontal (aphid and mechanical) and vertical (via tubers) transmission modes. Nucleotide diversities of viral populations were significantly influenced by transmission modes; lineages transmitted by aphids were the least diverse, whereas lineages transmitted by tubers were the most diverse. Differences in nucleotide diversities of viral populations between leaves and tubers were transmission mode-dependent, with higher diversities in tubers than in leaves for aphid and mechanically transmitted lineages. Furthermore, aphid and tuber transmissions were shown to impose stronger genetic bottlenecks than mechanical transmission. To better understand the structure of virus populations within the host, transmission mode, movement of the virus within the host, and the number of replication cycles after transmission event need to be considered. Collectively, our results suggest a significant impact of virus transmission modes on the within-plant diversity of virus populations and provide quantitative fundamental data for understanding how transmission can shape virus diversity in the natural ecosystems, where different transmission modes are expected to affect virus population structure and consequently its evolution.

Flooding-Associated Soft Rot of Sweetpotato Storage Roots Caused by Distinct Clostridium Isolates.

Flooding of sweetpotatoes in the field leads to development of soft rot on the storage roots while they remain submerged or on subsequent harvest and storage. Incidences of flooding after periods of intense rainy weather are on the rise in the southeastern United States, which is home to the majority of sweetpotato production in the nation. In an effort to characterize the causative agent(s) of this devastating disease, here we describe two distinct bacterial strains isolated from soft-rotted sweetpotato storage roots retrieved from an intentionally flooded field. Both of these anaerobic spore-forming isolates were identified as members of the genus Clostridium based on sequence similarity of multiple housekeeping genes, and both were confirmed to cause soft rot disease on sweetpotato and other vegetable crops. Despite these common features, the isolates were distinguishable by several phenotypic and biochemical properties, and phylogenetic analysis placed them in separate well-supported clades within the genus. Overall, our results demonstrate that multiple plant-pathogenic Clostridium species can cause soft rot disease on sweetpotato and suggest that a variety of other plant hosts may also be susceptible.

An aromatic amino acid and associated helix in the C-terminus of the potato leafroll virus minor capsid protein regulate systemic infection and symptom expression.

The C-terminal region of the minor structural protein of potato leafroll virus (PLRV), known as the readthrough protein (RTP), is involved in efficient virus movement, tissue tropism and symptom development. Analysis of numerous C-terminal deletions identified a five-amino acid motif that is required for RTP function. A PLRV mutant expressing RTP with these five amino acids deleted (Δ5aa-RTP) was compromised in systemic infection and symptom expression. Although the Δ5aa-RTP mutant was able to move long distance, limited infection foci were observed in systemically infected leaves suggesting that these five amino acids regulate virus phloem loading in the inoculated leaves and/or unloading into the systemically infected tissues. The 5aa deletion did not alter the efficiency of RTP translation, nor impair RTP self-interaction or its interaction with P17, the virus movement protein. However, the deletion did alter the subcellular localization of RTP. When co-expressed with a PLRV infectious clone, a GFP tagged wild-type RTP was localized to discontinuous punctate spots along the cell periphery and was associated with plasmodesmata, although localization was dependent upon the developmental stage of the plant tissue. In contrast, the Δ5aa-RTP-GFP aggregated in the cytoplasm. Structural modeling indicated that the 5aa deletion would be expected to perturb an α-helix motif. Two of 30 plants infected with Δ5aa-RTP developed a wild-type virus infection phenotype ten weeks post-inoculation. Analysis of the virus population in these plants by deep sequencing identified a duplication of sequences adjacent to the deletion that were predicted to restore the α-helix motif. The subcellular distribution of the RTP is regulated by the 5-aa motif which is under strong selection pressure and in turn contributes to the efficient long distance movement of the virus and the induction of systemic symptoms.

Genetic Variance Partitioning and Genome-Wide Prediction with Allele Dosage Information in Autotetraploid Potato.

As one of the world's most important food crops, the potato (Solanum tuberosum L.) has spurred innovation in autotetraploid genetics, including in the use of SNP arrays to determine allele dosage at thousands of markers. By combining genotype and pedigree information with phenotype data for economically important traits, the objectives of this study were to (1) partition the genetic variance into additive vs. nonadditive components, and (2) determine the accuracy of genome-wide prediction. Between 2012 and 2017, a training population of 571 clones was evaluated for total yield, specific gravity, and chip fry color. Genomic covariance matrices for additive (G), digenic dominant (D), and additive × additive epistatic (G#G) effects were calculated using 3895 markers, and the numerator relationship matrix (A) was calculated from a 13-generation pedigree. Based on model fit and prediction accuracy, mixed model analysis with G was superior to A for yield and fry color but not specific gravity. The amount of additive genetic variance captured by markers was 20% of the total genetic variance for specific gravity, compared to 45% for yield and fry color. Within the training population, including nonadditive effects improved accuracy and/or bias for all three traits when predicting total genotypic value. When six F1 populations were used for validation, prediction accuracy ranged from 0.06 to 0.63 and was consistently lower (0.13 on average) without allele dosage information. We conclude that genome-wide prediction is feasible in potato and that it will improve selection for breeding value given the substantial amount of nonadditive genetic variance in elite germplasm.