Four New Species of Small-Spored Alternaria Isolated from Solanum tuberosum and S. lycopersicum in China.

Small-spored Alternaria species have been frequently isolated from diseased leaves of Solanum plants. To clarify the diversity of small-spored Alternaria species, a total of 118 strains were obtained from leaf samples of S. tuberosum and S. lycopersicum in six provinces of China during 2022-2023. Based on morphological characterization and multi-locus phylogenetic analysis of the internal transcribed spacer of the rDNA region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1 alpha (TEF1), RNA polymerase second largest subunit (RPB2), Alternaria major allergen gene (Alt a 1), endopolygalacturonase gene (EndoPG) and an anonymous gene region (OPA10-2), seven species were determined, including four novel species and three known species (A. alternata, A. gossypina and A. arborescens). The novel species were described and illustrated as A. longxiensis sp. nov., A. lijiangensis sp. nov., A. lycopersici sp. nov. and A. solanicola sp. nov.. In addition, the pathogenicity of the seven species was evaluated on potato leaves. The species exhibited various aggressiveness, which could help in disease management.

Effects of different host plants on the growth, development, and fecundity of Phthorimaea absoluta (Lepidoptera: Gelechiidae): an evaluation based on the age-stage two-sex life table.

This study aimed to investigate the growth and development parameters of Phthorimaea absoluta (Meyrick) population at each stage when feeding on 4 host plants: Lycopersicon esculentum, Solanum tuberosum, Solanum melongena, and Nicotiana tabacum. The objective was to predict population dynamics and develop appropriate control strategies. The age-stage sex-life table was used to evaluate survival rate, fecundity, life expectancy, reproductive value, population parameters, and population growth prediction of P. absoluta after feeding on the 4 Solanaceae plants. The results showed significant variations in the fecundity parameters of P. absoluta among the different host plants. The L. esculentum population exhibited the highest average egg-laying period (13.17 ±â€…0.61 days) and average egg production (219.31 ±â€…21.02 eggs), while N. tabacum had the lowest values (4.56 ±â€…0.26 days and 26.08 ±â€…2.53 eggs, respectively). The gross reproduction rate of P. absoluta feeding on L. esculentum was 146.43 ±â€…21.00, which was 1.80, 3.77, and 6.39 times higher compared to S. tuberosum, S. melongena, and N. tabacum, respectively. The average age period and population doubling time of P. absoluta feeding on L. esculentum were lower than those of the other 3 host plants. These results indicated that while P. absoluta can complete a generation on L. esculentum, S. tuberosum, S. melongena, and N. tabacum, L. esculentum is the most suitable host for its growth and development. Therefore, in the occurrence and adjacent areas of P. absoluta, relevant authorities should promptly monitor and control its population in the planting areas of Solanaceae plants to prevent further spread.

CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato.

CRISPR/Cas9 technology has become the most efficient method for genome editing in many plant species, including important industrial crops such as potatoes. This study used three target regions (T1, T2, and T3) in gbss exon I, whose sequences were first inserted into the BbsI sites in the appropriate guide RNA (gRNA) vector (pEn-Chimera, pMR203, pMR204, and pMR205), and then localized between the AtU6 promoter and the gRNA scaffold sequence. Expression vectors were constructed by introducing gRNA genes into the pMR287 (pYUCas9Plus) plasmids using the MultiSite Gateway system by attR and attL sites. The three target regions of mutant potato lines were analyzed. The use of CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis allowed tri- or tetra-allelic mutant potato lines to be generated. Multiple nucleotide substitutions and indels within and around the three target sites caused a frameshift mutation that led to a premature stop codon, resulting in the production of gbss-knockout plants. Mutation frequencies and analysis of mutation patterns suggested that the stably transformed Cas9/multiple guide RNA expression constructs used in this study can induce targeted mutations efficiently in the potato genome. Full knockout of the gbss gene was analyzed by CAPS, Sanger sequencing and iodine staining. The present study demonstrated successful CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato gbss gene by Agrobacterium-mediated transformation, resulting in an amylose-free phenotype.

Genome-Wide Identification, Characterization and Expression Profiling of the CONSTANS-like Genes in Potato (Solanum tuberosum L.).

CONSTANS-like (COL) genes play important regulatory roles in flowering, tuber formation and the development of the potato (Solanum tuberosum L.). However, the COL gene family in S. tuberosum has not been systematically identified, restricting our knowledge of the function of these genes in S. tuberosum. In our study, we identified 14 COL genes, which were unequally distributed among eight chromosomes. These genes were classified into three groups based on differences in gene structure characteristics. The COL proteins of S. tuberosum and Solanum lycopersicum were closely related and showed high levels of similarity in a phylogenetic tree. Gene and protein structure analysis revealed similarities in the exon-intron structure and length, as well as the motif structure of COL proteins in the same subgroup. We identified 17 orthologous COL gene pairs between S. tuberosum and S. lycopersicum. Selection pressure analysis showed that the evolution rate of COL homologs is controlled by purification selection in Arabidopsis, S. tuberosum and S. lycopersicum. StCOL genes showed different tissue-specific expression patterns. StCOL5 and StCOL8 were highly expressed specifically in the leaves of plantlets. StCOL6, StCOL10 and StCOL14 were highly expressed in flowers. Tissue-specific expression characteristics suggest a functional differentiation of StCOL genes during evolution. Cis-element analysis revealed that the StCOL promoters contain several regulatory elements for hormone, light and stress signals. Our results provide a theoretical basis for the understanding of the in-depth mechanism of COL genes in regulating the flowering time and tuber development in S. tuberosum.

Genome-wide identification of 14-3-3 gene family and characterization of their expression in developmental stages of Solanum tuberosum under multiple biotic and abiotic stress conditions.

GF14 proteins are a family of conserved proteins involved in many cellular processes including transport, growth, metabolism, and stress response. However, only few reports are available regarding the 14-3-3 genes in potato. In this study, twelve 14-3-3 genes were detected in the potato genome. Based on their phylogenetic relationships, the StGF14 family members were categorized into two classes. Gene expression analysis demonstrated that StGF14h, StGF14a, and StGF14k had the highest gene expression, induced by abiotic and biotic stresses in all three tissues. The number of exons in 14-3-3 genes ranged from four to seven and most of these genes in the same subfamily had similar exon-intron patterns. The results of our study showed that the conserved motifs are similar in most of the proteins in each group. The intron-exon patterns and the composition of conserved motifs validated the 14-3-3 gene phylogenetic classification. According to the genome distribution results, 14-3-3 genes were located unevenly on the 12 Solanum tuberosum chromosomes. We find out 97 orthologous gene pairs between potato and Arabidopsis as well as 15 paralogous genes among potato genomes. Our results showed that GF-14 genes have an effective role in functional and molecular mechanisms in response to environmental stresses.

Qualitative and Quantitative Resistance against Early Blight Introgressed in Potato.

Early blight is a disease of potato that is caused by Alternaria species, notably A. solani. The disease is usually controlled with fungicides. However, A. solani is developing resistance against fungicides, and potato cultivars with genetic resistance to early blight are currently not available. Here, we identify two wild potato species, which are both crossable with cultivated potato (Solanum tuberosum), that show promising resistance against early blight disease. The cross between resistant S. berthaultii and a susceptible diploid S. tuberosum gave rise to a population in which resistance was inherited quantitatively. S. commersonii subsp. malmeanum was also crossed with diploid S. tuberosum, despite a differing endosperm balance number. This cross resulted in triploid progeny in which resistance was inherited dominantly. This is somewhat surprising, as resistance against necrotrophic plant pathogens is usually a quantitative trait or inherited recessively according to the inverse-gene-for-gene model. Hybrids with high levels of resistance to early blight are present among progeny from S. berthaultii as well as S. commersonii subsp. malmeanum, which is an important step towards the development of a cultivar with natural resistance to early blight.

Light Regulation of Chlorophyll and Glycoalkaloid Biosynthesis During Tuber Greening of Potato S. tuberosum.

Potato, S. tuberosum, is one of the most important global crops, but has high levels of waste due to tuber greening under light, which is associated with the accumulation of neurotoxic glycoalkaloids. However, unlike the situation in de-etiolating seedlings, the mechanisms underlying tuber greening are not well understood. Here, we have investigated the effect of monochromatic blue, red, and far-red light on the regulation of chlorophyll and glycoalkaloid accumulation in potato tubers. Blue and red wavelengths were effective for induction and accumulation of chlorophyll, carotenoids and the two major potato glycoalkaloids, α-solanine and α-chaconine, whereas none of these accumulated in darkness or under far-red light. Key genes in chlorophyll biosynthesis (HEMA1, encoding the rate-limiting enzyme glutamyl-tRNA reductase, GSA, CHLH and GUN4) and six genes (HMG1, SQS, CAS1, SSR2, SGT1 and SGT2) required for glycoalkaloid synthesis were also induced under white, blue, and red light but not in darkness or under far-red light. These data suggest a role for both cryptochrome and phytochrome photoreceptors in chlorophyll and glycoalkaloid accumulation. The contribution of phytochrome was further supported by the observation that far-red light could inhibit white light-induced chlorophyll and glycoalkaloid accumulation and associated gene expression. Transcriptomic analysis of tubers exposed to white, blue, and red light showed that light induction of photosynthesis and tetrapyrrole-related genes grouped into three distinct groups with one group showing a generally progressive induction by light at both 6 h and 24 h, a second group showing induction at 6 h in all light treatments, but induction only by red and white light at 24 h and a third showing just a very moderate light induction at 6 h which was reduced to the dark control level at 24 h. All glycoalkaloid synthesis genes showed a group one profile consistent with what was seen for the most light regulated chlorophyll synthesis genes. Our data provide a molecular framework for developing new approaches to reducing waste due to potato greening.

Interactive Responses of Potato (Solanum tuberosum L.) Plants to Heat Stress and Infection With Potato Virus Y.

Potato (Solanum tuberosum) plants are exposed to diverse environmental stresses, which may modulate plant-pathogen interactions, and potentially cause further decreases in crop productivity. To provide new insights into interactive molecular responses to heat stress combined with virus infection in potato, we analyzed expression of genes encoding pathogenesis-related (PR) proteins [markers of salicylic acid (SA)-mediated plant defense] and heat shock proteins (HSPs), in two potato cultivars that differ in tolerance to elevated temperatures and in susceptibility to potato virus Y (PVY). In plants of cv. Chicago (thermosensitive and PVY-susceptible), increased temperature reduced PR gene expression and this correlated with enhancement of PVY infection (virus accumulation and symptom production). In contrast, with cv. Gala (thermotolerant and PVY resistant), which displayed a greater increase in PR gene expression in response to PVY infection, temperature affected neither PR transcript levels nor virus accumulation. HSP genes were induced by elevated temperature in both cultivars but to higher levels in the thermotolerant (Gala) cultivar. PVY infection did not alter expression of HSP genes in the Gala cultivar (possibly because of the low level of virus accumulation) but did induce expression of HSP70 and HSP90 in the susceptible cultivar (Chicago). These findings suggest that responses to heat stress and PVY infection in potato have some common underlying mechanisms, which may be integrated in a specific consolidated network that controls plant sensitivity to multiple stresses in a cultivar-specific manner. We also found that the SA pre-treatment subverted the sensitive combined (heat and PVY) stress phenotype in Chicago, implicating SA as a key component of such a regulatory network.

Analysis of tail-anchored protein translocation pathway in plants.

Tail-anchored (TA) proteins are a special class of membrane proteins that carry out vital functions in all living cells. Targeting mechanisms of TA proteins are investigated as the best example for post-translational protein targeting in yeast. Of the several mechanisms, Guided Entry of Tail-anchored protein (GET) pathway plays a major role in TA protein targeting. Many in silico and in vivo analyses are geared to identify TA proteins and their targeting mechanisms in different systems including Arabidopsis thaliana. Yet, crop plants that grow in specific and/or different conditions are not investigated for the presence of TA proteins and GET pathway. This study majorly investigates GET pathway in two crop plants, Oryza sativa subsp. Indica and Solanum tuberosum, through detailed in silico analysis. 508 and 912 TA proteins are identified in Oryza sativa subsp. Indica and Solanum tuberosum respectively and their localization with respect to endoplasmic reticulum (ER), mitochondria, and chloroplast has been delineated. Similarly, the associated GET proteins are identified (Get1, Get3 and Get4) and their structural inferences are elucidated using homology modelling. Get3 models are based on yeast Get3. The cytoplasmic Get3 from O. sativa is identified to be very similar to yeast Get3 with conserved P-loop and TA binding groove. Three cytoplasmic Get3s are identified for S. tuberosum. Taken together, this is the first study to identify TA proteins and GET components in Oryza sativa subsp. Indica and Solanum tuberosum, forming the basis for any further experimental characterization of TA targeting and GET pathway mechanisms in crop plants.

Comparative genome analysis of Solanum lycopersicum and Solanum tuberosum.

Solanum lycopersicum and Solanum tuberosum are agriculturally important crop species as they are rich sources of starch, protein, antioxidants, lycopene, beta-carotene, vitamin C, and fiber. The genomes of S. lycopersicum and S. tuberosum are currently available. However the linear strings of nucleotides that together comprise a genome sequence are of limited significance by themselves. Computational and bioinformatics approaches can be used to exploit the genomes for fundamental research for improving their varieties. The comparative genome analysis, Pfam analysis of predicted reviewed paralogous proteins was performed. It was found that S. lycopersicum proteins belong to more families, domains and clans in comparison with S. tuberosum. It was also found that mostly intergenic regions are conserved in two genomes followed by exons, intron and UTR. This can be exploited to predict regions between genomes that are similar to each other and to study the evolutionary relationship between two genomes, leading towards the development of disease resistance, stress tolerance and improved varieties of tomato.

Interspecific somatic hybrids Solanum villosum (+) S. tuberosum, resistant to Phytophthora infestans.

The interspecific somatic hybrids 4x S. villosum (+) 2x S. tuberosum clone DG 81-68 (VT hybrids) were obtained and characterized molecularly and cytogenetically. The morphology of fusion-derived plants was intermediate in relation to the parental species. The expected ploidy level of the regenerants was 6x for the VT hybrids, but the real ploidy of the hybrids varied, with some of them being euploids, and others - aneuploids. The hybridity of the regenerants was verified by random amplified polymorphic DNA (RAPD) analysis. Despite the variation in ploidy, the RAPD patterns of the hybrids were mostly uniform, suggesting similarity of the genotypes of the VT clones. Genomic in situ hybridization (GISH) analysis discriminated between the chromosomes of both parental genomes in VT somatic hybrids and also confirmed their hybridity. The resistance of VT somatic hybrids to Phytophthora infestans was evaluated and all of the hybrids proved to be highly resistant. In search of the mechanisms involved in resistance of the Solanum species to P. infestans, the biochemical reactions occurring early after elicitor treatment were studied. The production of reactive oxygen species (ROS), as one of the earliest reactions induced by pathogens or their elicitors, was examined in the resistant wild species S. villosum, susceptible S. tuberosum clone DG 81-68 and in the VT hybrid, resistant to P. infestans. After treatment of the leaves with elicitor, the relative increase in ROS production was higher in leaves of the susceptible potato clone than in the resistant plants of S. villosum and the somatic hybrid.

EXPRESIÓN DE LA PROTEÍNA Cry1Ac EN TEJIDOS DE LÍNEASTRANSGÉNICAS DE PAPA (Solanum tuberosum spp. ANDÍGENA)VAR. DIACOL CAPIRO. / Cry1Ac Protein Expression in Tissues of Potato (Solanumtuberosum spp. Andígena) Transgenic Lines Var. Diacol Capiro

La papa (Solanum sp.) es el cuarto producto alimenticio más importante en el mundo. En Colombia anualmente se producen alrededor de 2,8 millones de toneladas, sirviendo como sustento económico a 90.000 familias. En el país, Tecia solanivora genera el mayor impacto económico en el cultivo con pérdidas de hasta el 100% en la producción de tubérculos. El fitomejoramiento vía introducción de genes Cry, que codifican para cristales proteicos insecticidas, constituye una alternativa para reducir el ataque de insectos en cultivos de interés comercial. En este trabajo se caracterizó la inserción, transcripción y expresión del gen Cry1Ac en diferentes tejidos y en tres etapas del desarrollo para dos líneas transgénicas de Solanumtuberosum spp. andígena variedad Diacol Capiro generadas previamente por transformación con Agrobacteriumtumefaciens. La caracterización se realizó a través de técnicas de PCR, RT-PCR y ELISA. Se corroboró la inserción y transcripción del gen utilizando primers que amplificaron una banda específica de 766pb para Cry1Ac. Los niveles de expresión de la proteína llegaron a ser mayores a 45µg/g y no mostraron diferencias significativas entre las líneas analizadas, ni entre las tres etapas del desarrollo. No se evidenciaron diferencias significativas entre las líneas transgénicas con respecto al control al hacer un análisis de algunas características fenotípicas relevantes. Los resultados encontrados sugieren la realización de seguimientos y ensayos de bioseguridad sobre este material, ya que los altos niveles de expresión en todos los tejidos analizados, pueden afectar a organismos no blanco.

The potato plant is the fourth most important crop in the world. In Colombia around 2.8 million tons are produced annually economically supporting 90000 families. In the country, the major economic impact in the crop is caused by Tecia solanivora that originates loses up to 100% in the tuber production. The genetic plant breeding related to the introduction of Cry genes which codify insecticidal crystal proteins is an alternative for reducing the insect attack in commercial crops. In this work, the insertion, transcription and expression of Cry1Ac gen was characterized in different tissues and three development stages of two transgenic lines of Solanum tuberosum variety Diacol Capiro that were previously transformed by Agrobacterium tumefaciens method. The characterization was realized by PCR, RT-PCR and ELISA techniques. The gen insertion and transcription was confirmed using primers for Cry1Ac gen that amplified a specific band of 766 bp. The protein expression levels were higher than 45 µg/g and were not significantly different between the analyzed lines nor the three development stages. Furthermore, taking into account some relevant phenotypic features, no significant differences were found between transgenic lines and controls. The results suggest that monitoring and biosecurity assays are necessary with this vegetal material because their high level expression inside all the tissues analyzed that could affect non-targeted insects.