Identification of potato varieties suitable for cold storage and reconditioning: A safer alternative to anti-sprouting chemicals for potato sprouting control.

Low temperature storage as an alternative to anti-sprouting chemicals in potato storage may induce reducing sugars (RS) accumulation (i.e. glucose and fructose) in potato tubers. This phenomenon is called "cold induced sweetening" (CIS) and occurs in certain varieties. CIS leads to a decrease in the organoleptic qualities and darkening of processed potato and the accumulation of toxic molecules such as acrylamide. To identify potato varieties suitable for storage at low temperatures, we screened six commercial processing varieties: Lady Claire (LC), Verdi, Kiebitz (KB), Pirol, Agria and Markies for their CIS characteristics and sprout-forming potential after storage at 4 °C and 8 °C. Our findings reveal that 4 °C storage allows for efficient sprout reduction in all six tested varieties for up to 4.5 months of storage. Three CIS-resistant varieties, namely Verdi, Lady Claire and Kiebitz, were identified as able to be stored for up to four months at 4 °C with limited increase in glucose content. Conversely, Pirol, Agria and Markies showed an increase in glucose content with a decrease in storage temperature and can be considered as CIS-susceptible varieties. After processing into crisps, the CIS-susceptible varieties displayed poor crisp color quality (brown to black color crisps) after storage for two months at 4 °C compared to the storage at 8 °C, whereas the CIS-resistant varieties had good crisp color quality (pale yellow color crisps) after storage at both 4 and 8 °C. Interestingly, the trends of total RS and/or glucose content in the CIS-resistant and in the CIS-susceptible varieties were correlated with the trends in Vacuolar Invertase (VInv) gene expression for most varieties, as well as with the trends in acrylamide content after processing. In addition, reconditioning of Markies variety after storage at 4 °C by gradually increasing the temperature to 15 °C resulted in a significant decrease of VInv transcript levels (reduction of 80 %), acrylamide content (reduction of 75 %) and glucose content when compared to a storage at 4 °C without reconditioning. Those results demonstrate that the reconditioning technique is a key factor for a sustainable potato storage and for improving the quality of processed potatoes.

Going beyond consensus genome sequences: An innovative SNP-based methodology reconstructs different Ugandan cassava brown streak virus haplotypes at a nationwide scale in Rwanda.

Cassava Brown Streak Disease (CBSD), which is caused by cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), represents one of the most devastating threats to cassava production in Africa, including in Rwanda where a dramatic epidemic in 2014 dropped cassava yield from 3.3 million to 900,000 tonnes (1). Studying viral genetic diversity at the genome level is essential in disease management, as it can provide valuable information on the origin and dynamics of epidemic events. To fill the current lack of genome-based diversity studies of UCBSV, we performed a nationwide survey of cassava ipomovirus genomic sequences in Rwanda by high-throughput sequencing (HTS) of pools of plants sampled from 130 cassava fields in thirteen cassava-producing districts, spanning seven agro-ecological zones with contrasting climatic conditions and different cassava cultivars. HTS allowed the assembly of a nearly complete consensus genome of UCBSV in twelve districts. The phylogenetic analysis revealed high homology between UCBSV genome sequences, with a maximum of 0.8 per cent divergence between genomes at the nucleotide level. An in-depth investigation based on Single Nucleotide Polymorphisms (SNPs) was conducted to explore the genome diversity beyond the consensus sequences. First, to ensure the validity of the result, a panel of SNPs was confirmed by independent reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. Furthermore, the combination of fixation index (FST) calculation and Principal Component Analysis (PCA) based on SNP patterns identified three different UCBSV haplotypes geographically clustered. The haplotype 2 (H2) was restricted to the central regions, where the NAROCAS 1 cultivar is predominantly farmed. RT-PCR and Sanger sequencing of individual NAROCAS1 plants confirmed their association with H2. Haplotype 1 was widely spread, with a 100 per cent occurrence in the Eastern region, while Haplotype 3 was only found in the Western region. These haplotypes' associations with specific cultivars or regions would need further confirmation. Our results prove that a much more complex picture of genetic diversity can be deciphered beyond the consensus sequences, with practical implications on virus epidemiology, evolution, and disease management. Our methodology proposes a high-resolution analysis of genome diversity beyond the consensus between and within samples. It can be used at various scales, from individual plants to pooled samples of virus-infected plants. Our findings also showed how subtle genetic differences could be informative on the potential impact of agricultural practices, as the presence and frequency of a virus haplotype could be correlated with the dissemination and adoption of improved cultivars.

An optimized hydroponic pipeline for large-scale identification of wheat genotypes with resilient biological nitrification inhibition activity.

Several plant species have been reported to inhibit nitrification via their root exudates, the so-called biological nitrification inhibition (BNI). Given the potential of BNI-producing plants to sustainably mitigate N losses in agrosystems, identification of BNI activity in existing germplasms is of paramount importance. A hydroponic system was combined with an optimized Nitrosomonas europaea-based bioassay to determine the BNI activity of root exudates. The pipeline allows collecting and processing hundreds of root exudates simultaneously. An additional assay was established to assess the potential bactericide effect of the root exudates. The pipeline was used to unravel the impact of developmental stage, temperature and osmotic stress on the BNI trait in selected wheat genotypes. Biological nitrification inhibition activity appeared consistently higher in wheat at the pretillering stage as compared to the tillering stage. While low-temperatures did not alter BNI activities in root exudates, osmotic stress appeared to change the BNI activity in a genotype-dependent manner. Further analysis of Nitrosomonas culture after pre-exposure to root exudates suggested that BNI activity has no or limited bactericide effects. The present pipeline will be instrumental to further investigating the dynamics of BNI activity and to uncover the diversity of the BNI trait in plant species.

A method for rapid and homogenous initiation of post-harvest physiological deterioration in cassava storage roots identifies Indonesian cultivars with improved shelf-life performance.

Cassava is the most cultivated and consumed root crop in the world. One of the major constraints to the cassava value chain is the short shelf life of cassava storage roots which is primarily due to the so-called post-harvest physiological deterioration (PPD). The identification of natural sources of PPD tolerance represents a key approach to mitigating PPD losses by generating farmer- and industry-preferred cassava cultivars with prolonged shelf life. In the present study, a PPD assessment method was developed to screen for PPD tolerance in the cassava germplasm. The proposed PPD assessment method displayed a reduced rate of microbial infection and allowed a rapid and homogenous development of typical PPD symptoms in the cassava storage roots. We successfully used the PPD assessment method in combination with an image-based PPD scoring method to identify and characterize PPD tolerance in 28 cassava cultivars from the Indonesian cassava germplasm. Our analysis showed a significant and positive correlation between PPD score and dry matter content (r = 0.589-0.664, p-value < 0.001). Analysis of additional root parameters showed a significant and positive correlation between PPD scores at 2 days post-harvest (dph) and root length (r = 0.388, p-value < 0.05). Our analysis identified at least 4 cultivars displaying a significantly delayed onset of PPD symptoms as compared to the other selected cultivars. The availability of cassava cultivars contrasting for tolerance to PPD will be particularly instrumental to understanding the molecular mechanisms associated with delayed PPD in cassava roots.

Harnessing plant viruses in the metagenomics era: from the development of infectious clones to applications.

Recent metagenomic studies which focused on virus characterization in the entire plant environment have revealed a remarkable viral diversity in plants. The exponential discovery of viruses also requires the concomitant implementation of high-throughput methods to perform their functional characterization. Despite several limitations, the development of viral infectious clones remains a method of choice to understand virus biology, their role in the phytobiome, and plant resilience. Here, we review the latest approaches for efficient characterization of plant viruses and technical advances built on high-throughput sequencing and synthetic biology to streamline assembly of viral infectious clones. We then discuss the applications of plant viral vectors in fundamental and applied plant research as well as their technical and regulatory limitations, and we propose strategies for their safer field applications.

Risk factors associated with cassava brown streak disease dissemination through seed pathways in Eastern D.R. Congo.

Vegetatively propagated crops are particularly prone to disease dissemination through their seed systems. Strict phytosanitary measures are important to limit the impact of diseases as illustrated by the potato seed system in Europe. Cassava brown streak disease (CBSD) is a devastating disease caused by two viral species collectively named cassava brown streak viruses (CBSVs). CBSD can cause substantial root yield losses of up to 100% in the worst affected areas and is easily transmitted through stem cuttings. In Eastern and Central Africa, the epidemiology of CBSVs in the local socio-economical context of production remains poorly known while a better understanding would be an asset to properly manage the disease. This lack of information explains partially the limited efficiency of current regulatory schemes in increasing the availability of quality seed to smallholders and mitigating the spread of pests and diseases. This study surveyed the epidemiology of CBSVs in Uvira territory, Eastern D.R. Congo, and its drivers using a multivariate approach combining farmer's interview, field observation, sampling and molecular detection of CBSVs. Investigation on the epidemiology of CBSD revealed that three clusters in the study area could be identified using five most significant factors: (i) symptoms incidence, (ii) number of whiteflies, (iii) types of foliar symptoms, (iv) cutting's pathways and (v) plant age. Among the three clusters identified, one proved to be potentially interesting for seed multiplication activities since the disease pressure was the lowest. Through risk assessment, we also identified several key socio-economic determinants on disease epidemy: (i) factors related to farmer's knowledge and awareness (knowledge of cassava pests and diseases, knowledge of management practices, support from extension services and management strategies applied), (ii) factors related to the geographical location of farmer's fields (proximity to borders, proximity to town, distance to acquire cuttings), as well as (iii) the pathways used to acquire cuttings.

Storage roots.

Vanderschuren and Agusti introduce plant storage roots.

Natural Variation in Vitamin B1 and Vitamin B6 Contents in Rice Germplasm.

Insufficient dietary intake of micronutrients contributes to the onset of deficiencies termed hidden hunger-a global health problem affecting approximately 2 billion people. Vitamin B1 (thiamine) and vitamin B6 (pyridoxine) are essential micronutrients because of their roles as enzymatic cofactors in all organisms. Metabolic engineering attempts to biofortify rice endosperm-a poor source of several micronutrients leading to deficiencies when consumed monotonously-have led to only minimal improvements in vitamin B1 and B6 contents. To determine if rice germplasm could be exploited for biofortification of rice endosperm, we screened 59 genetically diverse accessions under greenhouse conditions for variation in vitamin B1 and vitamin B6 contents across three tissue types (leaves, unpolished and polished grain). Accessions from low, intermediate and high vitamin categories that had similar vitamin levels in two greenhouse experiments were chosen for in-depth vitamer profiling and selected biosynthesis gene expression analyses. Vitamin B1 and B6 contents in polished seeds varied almost 4-fold. Genes encoding select vitamin B1 and B6 biosynthesis de novo enzymes (THIC for vitamin B1, PDX1.3a-c and PDX2 for vitamin B6) were differentially expressed in leaves across accessions contrasting in their respective vitamin contents. These expression levels did not correlate with leaf and unpolished seed vitamin contents, except for THIC expression in leaves that was positively correlated with total vitamin B1 contents in polished seeds. This study expands our knowledge of diversity in micronutrient traits in rice germplasm and provides insights into the expression of genes for vitamin B1 and B6 biosynthesis in rice.

Characterization of trichome-specific BAHD acyltransferases involved in acylsugar biosynthesis in Nicotiana tabacum.

Glandular trichomes of tobacco (Nicotiana tabacum) produce blends of acylsucroses that contribute to defence against pathogens and herbivorous insects, but the mechanism of assembly of these acylsugars has not yet been determined. In this study, we isolated and characterized two trichome-specific acylsugar acyltransferases that are localized in the endoplasmic reticulum, NtASAT1 and NtASAT2. They sequentially catalyse two additive steps of acyl donors to sucrose to produce di-acylsucrose. Knocking out of NtASAT1 or NtASAT2 resulted in deficiency of acylsucrose; however, there was no effect on acylsugar accumulation in plants overexpressing NtASAT1 or NtASAT2. Genomic analysis and profiling revealed that NtASATs originated from the T subgenome, which is derived from the acylsugar-producing diploid ancestor N. tomentosiformis. Our identification of NtASAT1 and NtASAT2 as enzymes involved in acylsugar assembly in tobacco potentially provides a new approach and target genes for improving crop resistance against pathogens and insects.

Contamination in Reference Sequence Databases: Time for Divide-and-Rule Tactics.

Contaminating sequences in public genome databases is a pervasive issue with potentially far-reaching consequences. This problem has attracted much attention in the recent literature and many different tools are now available to detect contaminants. Although these methods are based on diverse algorithms that can sometimes produce widely different estimates of the contamination level, the majority of genomic studies rely on a single method of detection, which represents a risk of systematic error. In this work, we used two orthogonal methods to assess the level of contamination among National Center for Biotechnological Information Reference Sequence Database (RefSeq) bacterial genomes. First, we applied the most popular solution, CheckM, which is based on gene markers. We then complemented this approach by a genome-wide method, termed Physeter, which now implements a k-folds algorithm to avoid inaccurate detection due to potential contamination of the reference database. We demonstrate that CheckM cannot currently be applied to all available genomes and bacterial groups. While it performed well on the majority of RefSeq genomes, it produced dubious results for 12,326 organisms. Among those, Physeter identified 239 contaminated genomes that had been missed by CheckM. In conclusion, we emphasize the importance of using multiple methods of detection while providing an upgrade of our own detection tool, Physeter, which minimizes incorrect contamination estimates in the context of unavoidably contaminated reference databases.

Novel Ampeloviruses Infecting Cassava in Central Africa and the South-West Indian Ocean Islands.

Cassava is one of the most important staple crops in Africa and its production is seriously damaged by viral diseases. In this study, we identify for the first time and characterize the genome organization of novel ampeloviruses infecting cassava plants in diverse geographical locations using three high-throughput sequencing protocols [Virion-Associated Nucleotide Acid (VANA), dsRNA and total RNA], and we provide a first analysis of the diversity of these agents and of the evolutionary forces acting on them. Thirteen new Closteroviridae isolates were characterized in field-grown cassava plants from the Democratic Republic of Congo (DR Congo), Madagascar, Mayotte, and Reunion islands. The analysis of the sequences of the corresponding contigs (ranging between 10,417 and 13,752 nucleotides in length) revealed seven open reading frames. The replication-associated polyproteins have three expected functional domains: methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp). Additional open reading frames code for a small transmembrane protein, a heat-shock protein 70 homolog (HSP70h), a heat shock protein 90 homolog (HSP90h), and a major and a minor coat protein (CP and CPd respectively). Defective genomic variants were also identified in some cassava accessions originating from Madagascar and Reunion. The isolates were found to belong to two species tentatively named Manihot esculenta-associated virus 1 and 2 (MEaV-1 and MEaV-2). Phylogenetic analyses showed that MEaV-1 and MEaV-2 belong to the genus Ampelovirus, in particular to its subgroup II. MEaV-1 was found in all of the countries of study, while MEaV-2 was only detected in Madagascar and Mayotte. Recombination analysis provided evidence of intraspecies recombination occurring between the isolates from Madagascar and Mayotte. No clear association with visual symptoms in the cassava host could be identified.

Efficient Genetic Transformation and Regeneration of a Farmer-Preferred Cassava Cultivar From Ghana.

Cassava is an important staple crop that provides food and income for about 700 million Africans. Cassava productivity in Africa is limited by viral diseases, mainly cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Genetic barriers such as high heterozygosity, allopolyploidy, poor seed set, and irregular flowering constrain the development of virus-resistant cassava varieties via conventional breeding. Genetic transformation represents a valuable tool to circumvent several challenges associated with the development of virus resistance and other valuable agronomic traits in cassava. The implementation of genetic transformation in many local African cultivars is limited either by the difficulty to produce friable embryogenic callus (FEC), low transformation, and/or regeneration efficiencies. Here, we report the successful induction of organized embryogenic structures (OES) in 11 farmer-preferred cultivars locally grown in Ghana. The production of high quality FEC from one local cultivar, ADI 001, facilitated its genetic transformation with high shoot regeneration and selection efficiency, comparable to the model cassava cultivar 60444. We show that using flow cytometry for analysis of nuclear ploidy in FEC tissues prior to genetic transformation ensures the selection of genetically uniform FEC tissue for transformation. The high percentage of single insertion events in transgenic lines indicates the suitability of the ADI 001 cultivar for the introduction of virus resistance and other useful agronomic traits into the farmer-preferred cassava germplasm in Ghana and Africa.

Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants.

To meet increasing global food demand, breeders and scientists aim to improve the yield and quality of major food crops. Plant diseases threaten food security and are expected to increase because of climate change. CRISPR genome-editing technology opens new opportunities to engineer disease resistance traits. With precise genome engineering and transgene-free applications, CRISPR is expected to resolve the major challenges to crop improvement. Here, we discuss the latest developments in CRISPR technologies for engineering resistance to viruses, bacteria, fungi, and pests. We conclude by highlighting current concerns and gaps in technology, as well as outstanding questions for future research.

Multiplying the efficiency and impact of biofortification through metabolic engineering.

Ending all forms of hunger by 2030, as set forward in the UN-Sustainable Development Goal 2 (UN-SDG2), is a daunting but essential task, given the limited timeline ahead and the negative global health and socio-economic impact of hunger. Malnutrition or hidden hunger due to micronutrient deficiencies affects about one third of the world population and severely jeopardizes economic development. Staple crop biofortification through gene stacking, using a rational combination of conventional breeding and metabolic engineering strategies, should enable a leap forward within the coming decade. A number of specific actions and policy interventions are proposed to reach this goal.

Screening for Resistance in Farmer-Preferred Cassava Cultivars from Ghana to a Mixed Infection of CBSV and UCBSV.

Cassava brown streak disease (CBSD) caused by the Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is a threat to cassava production in Africa. The potential spread of CBSD into West Africa is a cause for concern, therefore screening for resistance in farmer-preferred genotypes is crucial for effective control and management. We multiplied a selection of eleven cassava cultivars grown by farmers in Ghana to test their response to a mixed infection of CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02) isolates using a stringent top-cleft graft inoculation method. Virus titers were quantified in the inoculated scions and cuttings propagated from the inoculated scions to assess virus accumulation and recovery. All cultivars were susceptible to the mixed infection although their response and symptom development varied. In the propagated infected scions, CBSV accumulated at higher titers in leaves of eight of the eleven cultivars. Visual scoring of storage roots from six-month-old virus-inoculated plants revealed the absence of CBSD-associated necrosis symptoms and detectable titers of CBSVs in the cultivar, IFAD. Although all eleven cultivars supported the replication of CBSV and UCBSV in their leaves, the absence of virus replication and CBSD-associated symptoms in the roots of some cultivars could be used as criteria to rapidly advance durable CBSD tolerance using breeding and genetic engineering approaches.

Asparagine accumulation in chicory storage roots is controlled by translocation and feedback regulation of asparagine biosynthesis in leaves.

The presence of acrylamide (AA), a potentially carcinogenic and neurotoxic compound, in food has become a major concern for public health. AA in plant-derived food mainly arises from the reaction of the amino acid asparagine (Asn) and reducing sugars during processing of foodstuffs at high temperature. Using a selection of genotypes from the chicory (Cichorium intybus L.) germplasm, we performed Asn measurements in storage roots and leaves to identify genotypes contrasting for Asn accumulation. We combined molecular analysis and grafting experiments to show that leaf to root translocation controls Asn biosynthesis and accumulation in chicory storage roots. We could demonstrate that Asn accumulation in storage roots depends on Asn biosynthesis and transport from the leaf, and that a negative feedback loop by Asn on CiASN1 expression impacts Asn biosynthesis in leaves. Our results provide a new model for Asn biosynthesis in root crop species and highlight the importance of characterizing and manipulating Asn transport to reduce AA content in processed plant-based foodstuffs.

Virus-Induced Gene Silencing (VIGS) in Cassava Using Geminivirus Agroclones.

Virus-induced gene silencing (VIGS) is an efficient, low-cost, and rapid functional validation tool for candidate genes in planta. The VIGS approach is particularly suitable to perform reverse genetics studies in crop species. Here we present a detailed method to perform VIGS in cassava, from target gene fragment to agroinoculation and VIGS quantitation.