Introduction and scientific justification of data transportability for confined field testing for the ERA of GM plants.

The concept of Data Transportability (DT) of Confined Field Testing (CFT) to support the Environmental Risk Assessment (ERA) of Genetically Modified (GM) plants was first introduced in the literature by Garcia-Alonso et al., in 2014. Since then, DT has been discussed in many countries and regions as a concept to prevent duplication of regulatory studies without compromising quality of the ERA. However, despite its usefulness and scientific justification, DT is not well adopted at this time and many regulatory agencies around the world require additional in-country CFT be conducted before approving GM plants. Based on the current circumstances, the authors organized a parallel session entitled "Introduction and Scientific Justification of DT for CFT for the ERA of GM plants" at 16th ISBR (the International Society for Biosafety Research). This session mainly consisted of the following three parts. The first two speakers, Andrew Roberts and Abigail Simmons provided an overview of DT and examples of conditions for the transportability of field data/conclusions advocated in the peer-reviewed scientific journals. Next, the current status of DT adoption in some countries/regions such as Japan and Africa, and a theoretical case study for Argentina were introduced by Kazuyuki Hiratsuka, Douglas Miano, and Facundo Vesprini, respectively. Lastly, a risk hypothesis-based approach for DT which was developed in advance by the five speakers of this parallel session, was introduced. During the discussion, there was a common understanding that transition to the risk hypothesis-based approach for DT was scientifically appropriate, considering the accumulated evidences that several countries have conducted confirmatory local CFT for more than 20 years but they have not detected any differences related to the ERA assessment endpoints in GM crops. The risk hypothesis-based approach for DT introduced here is expected to play an important role in discussions on the implementation of DT in various parts of the world in the future.

Biocontrol-based strategies for improving soil health and managing plant-parasitic nematodes in coffee production.

Coffee is an important commodity for Kenya, where production is steadily declining, despite a global rise in demand. Of the various constraints affecting production, plant-parasitic nematodes are a significant, but often overlooked, threat. As a perennial crop, treating plantations once infected with nematodes becomes difficult. The current study evaluated the drenching application of two biocontrol agents, Trichoderma asperellum and Purpureocillium lilacinum, for their nematode control efficacy, as well as their impact on the soil nematode community structure on mature, established coffee trees in Kenya. Seven Arabica coffee field trials were conducted over two years on trees of various ages. All the fields were heavily infested with Meloidogyne hapla, the first report of the species on coffee in Kenya. Both fungal biocontrol agents were detected endophytically infecting roots and recovered from soil but not until six months after initial applications. The population densities of M. hapla had significantly declined in roots of treated trees 12 months after the initial application, although soil nematode density data were similar across treatments. Based upon the maturity index and the Shannon index, treatment with T. asperellum led to improved soil health conditions and enrichment of diversity in the microbial community. Application of P. lilacinum, in particular, led to an increased abundance of fungivorous nematodes, especially Aphelenchus spp., for which P. lilacinum would appear to be a preferred food source. The soils in the trials were all stressed and denuded, however, which likely delayed the impact of such treatments or detection of any differences between treatments using indices, such as the functional metabolic footprint, over the period of study. A longer period of study would therefore likely provide a better indication of treatment benefits. The current study positively demonstrates, however, the potential for using biologically based options for the environmentally and climate-smart management of nematode threats in a sustainable manner on established, mature coffee plantations.

Survey for Virus Diversity in Common Bean (Phaseolus vulgaris) Fields and the Detection of a Novel Strain of Cowpea polerovirus 1 in Zambia.

The production of common bean (Phaseolus vulgaris L.) is adversely affected by virus-like diseases globally, but little is known about the occurrence, distribution, and diversity of common bean-infecting viruses in Zambia. Consequently, field surveys were conducted during the 2018 season in 128 fields across six provinces of Zambia and 640 common bean leaf tissue samples were collected with (n = 585) or without (n = 55) symptoms. The prevalence of symptomatic fields was 100%, but incidence of symptomatic plants ranged from 32 to 67.5%. Metagenomic analyses of nine composite samples and a single plant sample of interest revealed the occurrence of isolates of Bean common mosaic necrosis virus, Bean common mosaic virus, Cowpea aphid-borne mosaic virus, Peanut mottle virus, Southern bean mosaic virus (SBMV), Cucumber mosaic virus, Phaseolus vulgaris alphaendornavirus 1 (PvEV-1), PvEV-2, Ethiopian tobacco bushy top virus (ETBTV), and a novel strain of Cowpea polerovirus 1 (CPPV1-Pv) of 5,902 nt in length. While CPPV1-Pv was consistently detected in mixed infection with ETBTV and its satellite RNA molecule, based on results of mechanical transmission assays it does not appear to be involved in disease etiology, suggesting that its role may be limited to being a helper virus for the umbravirus. Screening of the survey samples by real-time PCR for the viruses detected by high-throughput sequencing revealed the prevalence of single (65.2% or 417/640) over mixed (1.9% or 12/640) infections in the samples. SBMV was the most frequently detected virus, occurring in ∼29.4% (188/640) of the samples and at a prevalence rate of 58.6% (75/128) across fields. The results showed that diverse virus species are present in Zambian common bean fields and the information will be useful for the management of common bean viral diseases.

Identification of an Elite Core Panel as a Key Breeding Resource to Accelerate the Rate of Genetic Improvement for Irrigated Rice.

Rice genetic improvement is a key component of achieving and maintaining food security in Asia and Africa in the face of growing populations and climate change. In this effort, the International Rice Research Institute (IRRI) continues to play a critical role in creating and disseminating rice varieties with higher productivity. Due to increasing demand for rice, especially in Africa, there is a strong need to accelerate the rate of genetic improvement for grain yield. In an effort to identify and characterize the elite breeding pool of IRRI's irrigated rice breeding program, we analyzed 102 historical yield trials conducted in the Philippines during the period 2012-2016 and representing 15,286 breeding lines (including released varieties). A mixed model approach based on the pedigree relationship matrix was used to estimate breeding values for grain yield, which ranged from 2.12 to 6.27 t·ha-1. The rate of genetic gain for grain yield was estimated at 8.75 kg·ha-1 year-1 (0.23%) for crosses made in the period from 1964 to 2014. Reducing the data to only IRRI released varieties, the rate doubled to 17.36 kg·ha-1 year-1 (0.46%). Regressed against breeding cycle the rate of gain for grain yield was 185 kg·ha-1 cycle-1 (4.95%). We selected 72 top performing lines based on breeding values for grain yield to create an elite core panel (ECP) representing the genetic diversity in the breeding program with the highest heritable yield values from which new products can be derived. The ECP closely aligns with the indica 1B sub-group of Oryza sativa that includes most modern varieties for irrigated systems. Agronomic performance of the ECP under multiple environments in Asia and Africa confirmed its high yield potential. We found that the rate of genetic gain for grain yield found in this study was limited primarily by long cycle times and the direct introduction of non-improved material into the elite pool. Consequently, the current breeding scheme for irrigated rice at IRRI is based on rapid recurrent selection among highly elite lines. In this context, the ECP constitutes an important resource for IRRI and NAREs breeders to carefully characterize and manage that elite diversity.

Loop-Mediated Isothermal Amplification assays for on-site detection of the main sweetpotato infecting viruses.

Globally, Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV) occur frequently and in combination cause sweetpotato virus disease (SPVD). Many viral diseases are economically important and negatively impact the production and movement of germplasm across regions. Rapid detection of viruses is critical for effective control. Detection and quantification of viruses directly from sweetpotato remains a challenge. Current diagnostic tests are not sensitive enough to reliably detect viruses directly from the plant or require expensive laboratory equipment and expertise to perform. We developed a simple and rapid loop-mediated isothermal amplification (LAMP) assay for the detection of SPFMV, SPCSV and begomoviruses related to sweet potato leaf curl virus (SPLCV). Laboratory validation recorded 100 % diagnostic sensitivity for all the three viruses. The LAMP assays were customized for field testing using a lyophilized thermostable isothermal master mix in a ready-to-use form that required no cold chain. The average time to positivity (TTP) was: SPFMV 5-30 min, SPCSV 15-43 min s and begomoviruses 28-45 mins. LAMP on-site testing results were comparable to PCR and RT-PCR confirmatory laboratory tests. The LAMP assay is a powerful tool for rapid sweetpotato virus detection at a reasonable cost and thus could serve as quality control systems for planting materials.

First report of Ethiopian tobacco bushy top virus and its associated satellite RNA infecting common bean (Phaseolus vulgaris L.) in Zambia.

During surveys for common bean viruses in Central Province of Zambia in April 2018, symptoms of bushy top, deep green curled branches and patchy leaf chlorosis were observed on five plants in a 2-ha farmer's field. Total RNA was isolated from symptomatic leaf samples using the CTAB method (Chang et al. 1993). The RNA from one sample (CP414-1) was used to construct a cDNA library with the Illumina TruSeq RNA Library Prep Kit (Illumina, San Diego, CA), followed by high-throughput sequencing (HTS) on the Illumina MiSeq platform that generated ~3.1M single-end raw reads of ~300 nucleotides (nt) each. A total of 355,885 reads showed hits to Ethiopian tobacco bushy top virus (ETBTV; Umbravirus), ETBTV satellite RNA (satRNA) and peanut mottle virus (PeMoV, Potyvirus) based on BLASTn analysis. The full-length genomes of ETBTV (4239-nt; MT225089), its satRNA (521-nt; MT225092) and PeMoV (9,643-nt) were assembled from the HTS reads using Geneious R11.1.2 (Biomatters, Auckland, New Zealand). The obtained complete genome sequences of ETBTV (MT225089) and ETBTV satRNA (MT225092) shared 88% and 95% nt identities, respectively with the corresponding viral (KJ918748) and satRNA (KJ918747) sequences of isolate 18-2 (Abraham et al. 2014). The near complete PeMoV genome was 89% identical to isolate Liaoning (MH270528). The HTS results were validated by two-step RT-PCR analyses of the five field-collected samples using newly designed primer pairs (data not shown). All five samples gave the expected 988-bp ETBTV-specific and 521-bp satRNA-specific DNA bands while three samples produced the expected 2100-bp PeMoV-specific fragment. The virus specificities of the agent specific PCR fragments were ascertained by Sanger sequencing (ETBTV: MT225090-91; ETBTV satRNA: MT225093-94; PeMoV: MT900843-44) and they shared 98-100% identities with their corresponding HTS-derived sequences. To further probe for the presence of an ETBTV helper virus, the samples were screened by RT-PCR with the degenerate primer pair Lu1-mod-F/C2R3 that was modified from Robertson et al. (1991). The expected 245-bp DNA bands was obtained from all five samples, indicating the presence of a possible luteovirus or polerovirus target in these samples. The BLASTn analyses of the two Sanger sequenced gel-eluted products (MT900845-46) showed that they shared 100% identity with each other and 96% nt identity with cowpea polerovirus 1 (CPPV1, KX599163). Leaf tissue extracts from a common bean plant that was confirmed by RT-PCR to be positive for all four agents were rub-inoculated onto Nicotiana occidentalis and common bean (Sutter Pink) plants (n=5 each) at the three fully expanded leaf stage, with a buffer inoculation as control. Systemic foliar symptoms consisting of leaf deformation, stunting and leaf bushy top were observed on all ten plants, 10 days post-inoculation whereas the control plants remained symptomless. All the test plants were screened by RT-PCR as described above. The results showed that all five N. occidentalis plants were positive for ETBTV+ETBTVsatRNA, the five common bean plants tested positive for ETBTV+satRNA+PeMoV, and all 10 plants of both species were negative for CPPV1. To the best of our knowledge, this is the first report of ETBTV, ETBTV satRNA and CPPV1 infecting common bean in Zambia, and the first molecular based confirmation of PeMoV occurrence in the country. Ongoing studies are focused on determining the extent of the disease spread and assessment of its economic impact.

Storage Root Yield of Sweetpotato as Influenced by Sweetpotato leaf curl virus and Its Interaction With Sweetpotato feathery mottle virus and Sweetpotato chlorotic stunt virus in Kenya.

In this study, the effect of a Kenyan strain of Sweetpotato leaf curl virus (SPLCV) and its interactions with Sweetpotato feathery mottle virus (SPFMV) and Sweetpotato chlorotic stunt virus (SPCSV) on root yield was determined. Trials were performed during two seasons using varieties Kakamega and Ejumula and contrasting in their resistance to sweetpotato virus disease in a randomized complete block design with 16 treatments replicated three times. The treatments included plants graft inoculated with SPLCV, SPFMV, and SPCSV alone and in possible dual or triple combinations. Yield and yield-related parameters were evaluated at harvest. The results showed marked differences in the effect of SPLCV infection on the two varieties. Ejumula, which is highly susceptible to SPFMV and SPCSV, suffered no significant yield loss from SPLCV infection, whereas Kakamega, which is moderately resistant to SPFMV and SPCSV, suffered an average of 47% yield loss from SPLCV, despite only mild symptoms occurring in both varieties. These results highlight the variability in yield response to SPLCV between sweetpotato cultivars as well as a lack of correlation of SPLCV-related symptoms with yield reduction. In addition, they underline the lack of correlation between resistance to the RNA viruses SPCSV and SPFMV and the DNA virus SPLCV.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Immunohistochemical localization of Cassava brown streak virus and its morphological effect on cassava leaves.

The localization of Cassava brown streak virus (CBSV) in cassava (Manihot esculenta) leaf tissues was determined and cellular morphological changes in CBSV-infected tissues were evaluated. CBSV-symptomatic leaves were screened with CBSV-specific primers using reverse-transcriptase polymerase chain reaction. Immunohistochemical reactions showed precipitation in CBSV-infected but not CBSV-free tissues, demonstrating successful localization of CBSV. Microscopic inspection showed significantly larger (P < 0.001) midribs in CBSV-infected compared with control (uninfected) leaves. Viral accumulation occurred in middle and lower but rarely in young upper leaves. This immunohistochemical method for virus localization will be invaluable for efficient screening of CBSV and for breeding resistant cassava.

Differential response of cassava genotypes to infection by cassava mosaic geminiviruses.

Mitigation of cassava mosaic disease (CMD) focuses on the introgression of resistance imparted by the polygenic recessive (CMD1), dominant monogenic (CMD2) and CMD3 loci. The mechanism(s) of resistance they impart, however, remain unknown. Two CMD susceptible and nine CMD resistant cassava genotypes were inoculated by microparticle bombardment with infectious clones of African cassava mosaic virus Cameroon strain (ACMV-CM) and the Kenyan strain K201 of East African cassava mosaic virus (EACMV KE2 [K201]). Genotypes carrying the CMD1 (TMS 30572), CMD2 (TME 3, TME 204 and Oko-iyawo) and CMD3 (TMS 97/0505) resistance mechanisms showed high levels of resistance to ACMV-CM, with viral DNA undetectable by PCR beyond 7days post inoculation (dpi). In contrast, all genotypes initially developed severe CMD symptoms and accumulated high virus titers after inoculation with EACMV KE2 (K201). Resistant genotypes recovered to become asymptomatic by 65dpi with no detectable virus in newly formed leaves. Genotype TMS 97/2205 showed highest resistance to EACMV KE2 (K201) with <30% of inoculated plants developing symptoms followed by complete recovery by 35dpi. Deep sequencing of small RNAs confirmed production of 21-24 nt virus derived small RNAs (vsRNA) that mapped to cover the entire ACMV-CM and EACMV KE2 (K201) viral genomes in both polarities, with hotspots seen within gene coding regions. In resistant genotypes, total vsRNAs were most abundant at 20 and 35dpi but reduced significantly upon recovery from CMD. In contrast, CMD susceptible genotypes displayed abundant vsRNAs throughout the experimental period. The percentage of vsRNAs reads ranked by class size were 21nt (45%), 22 nt (28%) and 24 nt (18%) in all genotypes studied. The number of vsRNA reads directly correlated with virus titer and CMD symptoms.

Field Level RNAi-Mediated Resistance to Cassava Brown Streak Disease across Multiple Cropping Cycles and Diverse East African Agro-Ecological Locations.

Cassava brown streak disease (CBSD) presents a serious threat to cassava production in East and Central Africa. Currently, no cultivars with high levels of resistance to CBSD are available to farmers. Transgenic RNAi technology was employed to combat CBSD by fusing coat protein (CP) sequences from Ugandan cassava brown streak virus (UCBSV) and Cassava brown streak virus (CBSV) to create an inverted repeat construct (p5001) driven by the constitutive Cassava vein mosaic virus promoter. Twenty-five plant lines of cultivar TME 204 expressing varying levels of small interfering RNAs (siRNAs) were established in confined field trials (CFTs) in Uganda and Kenya. Within an initial CFT at Namulonge, Uganda, non-transgenic TME 204 plants developed foliar and storage root CBSD incidences at 96-100% by 12 months after planting. In contrast, 16 of the 25 p5001 transgenic lines showed no foliar symptoms and had less than 8% of their storage roots symptomatic for CBSD. A direct positive correlation was seen between levels of resistance to CBSD and expression of transgenic CP-derived siRNAs. A subsequent CFT was established at Namulonge using stem cuttings from the initial trial. All transgenic lines established remained asymptomatic for CBSD, while 98% of the non-transgenic TME 204 stake-derived plants developed storage roots symptomatic for CBSD. Similarly, very high levels of resistance to CBSD were demonstrated by TME 204 p5001 RNAi lines grown within a CFT over a full cropping cycle at Mtwapa, coastal Kenya. Sequence analysis of CBSD causal viruses present at the trial sites showed that the transgenic lines were exposed to both CBSV and UCBSV, and that the sequenced isolates shared >90% CP identity with transgenic CP sequences expressed by the p5001 inverted repeat expression cassette. These results demonstrate very high levels of field resistance to CBSD conferred by the p5001 RNAi construct at diverse agro-ecological locations, and across the vegetative cropping cycle.

A Virus-Derived Stacked RNAi Construct Confers Robust Resistance to Cassava Brown Streak Disease.

Cassava brown streak disease (CBSD) threatens food and economic security for smallholder farmers throughout East and Central Africa, and poses a threat to cassava production in West Africa. CBSD is caused by two whitefly-transmitted virus species: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) (Genus: Ipomovirus, Family Potyviridae). Although varying levels of tolerance have been achieved through conventional breeding, to date, effective resistance to CBSD within East African cassava germplasm has not been identified. RNAi technology was utilized to integrate CBSD resistance into the Ugandan farmer-preferred cassava cultivar TME 204. Transgenic plant lines were generated expressing an inverted repeat construct (p5001) derived from coat-protein (CP) sequences of CBSV and UCBSV fused in tandem. Northern blots using probes specific for each CP sequence were performed to characterize 169 independent transgenic lines for accumulation of CP-derived siRNAs. Transgenic plant lines accumulating low, medium and high levels of siRNAs were bud graft challenged with the virulent CBSV Naliendele isolate alone or in combination with UCBSV. Resistance to CBSD in the greenhouse directly correlated to levels of CP-derived siRNAs as determined by visual assessment of leaf and storage root symptoms, and RT-PCR diagnosis for presence of the pathogens. Low expressing lines were found to be susceptible to CBSV and UCBSV, while medium to high accumulating plant lines were resistant to both virus species. Absence of detectable virus in the best performing p5001 transgenic lines was further confirmed by back-inoculation via sap or graft challenge to CBSD susceptible Nicotiana benthamiana and cassava cultivar 60444, respectively. Data presented shows robust resistance of transgenic p5001 TME 204 lines to both CBSV and UCBSV under greenhouse conditions. Levels of resistance correlated directly with levels of transgene derived siRNA expression such that the latter can be used as predictor of resistance to CBSD.

Loss of CMD2-mediated resistance to cassava mosaic disease in plants regenerated through somatic embryogenesis.

Cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are the two most important viral diseases affecting cassava production in Africa. Three sources of resistance are employed to combat CMD: polygenic recessive resistance, termed CMD1, the dominant monogenic type, named CMD2, and the recently characterized CMD3. The farmer-preferred cultivar TME 204 carries inherent resistance to CMD mediated by CMD2, but is highly susceptible to CBSD. Selected plants of TME 204 produced for RNA interference (RNAi)-mediated resistance to CBSD were regenerated via somatic embryogenesis and tested in confined field trials in East Africa. Although micropropagated, wild-type TME 204 plants exhibited the expected levels of resistance, all plants regenerated via somatic embryogenesis were found to be highly susceptible to CMD. Glasshouse studies using infectious clones of East African cassava mosaic virus conclusively demonstrated that the process of somatic embryogenesis used to regenerate cassava caused the resulting plants to become susceptible to CMD. This phenomenon could be replicated in the two additional CMD2-type varieties TME 3 and TME 7, but the CMD1-type cultivar TMS 30572 and the CMD3-type cultivar TMS 98/0505 maintained resistance to CMD after passage through somatic embryogenesis. Data are presented to define the specific tissue culture step at which the loss of CMD resistance occurs and to show that the loss of CMD2-mediated resistance is maintained across vegetative generations. These findings reveal new aspects of the widely used technique of somatic embryogenesis, and the stability of field-level resistance in CMD2-type cultivars presently grown by farmers in East Africa, where CMD pressure is high.

Survey, Molecular Detection, and Characterization of Geminiviruses Associated with Cassava Mosaic Disease in Zambia.

A survey was conducted from April to May 2014 in 214 farmers' fields located across six major cassava-producing provinces (Western, Northwestern, Northern, Luapula, Lusaka, and Eastern) of Zambia to determine the status of cassava mosaic disease (CMD) and the species diversity of associated cassava mosaic geminiviruses (CMG). Mean CMD incidence varied across all six provinces but was greatest in Lusaka Province (81%) and least in Northern Province (44%). Mean CMD severity varied slightly between provinces, ranging from 2.78 in Eastern Province to 3.00 in Northwestern Province. Polymerase chain reaction discrimination of 226 survey samples, coupled with complete DNA-A genome sequence analysis, revealed the presence of African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), and East African cassava mosaic Malawi virus (EACMMV) as single or mixed infections of different proportions. Single-virus infections were predominant, occurring in 62.8% (ACMV), 5.8% (EACMMV), and 2.2% (EACMV) of samples relative to mixed-virus infections, which occurred in 19.5% (ACMV + EACMMV), 0.4% (ACMV + EACMV), and 0.9% (ACMV + EACMV + EACMMV) of samples. Phylogenetic analysis revealed the segregation of virus isolates from Zambia into clades specific to ACMV, EACMV, and EACMMV, further confirming the presence of all three viruses in Zambia. The results point to a greater diversity of CMG across major cassava-growing provinces of Zambia and implicate contaminated cassava cuttings in disease spread.

The VIRCA Project: virus resistant cassava for Africa.

The VIRCA (Virus Resistant Cassava for Africa) project is a collaborative program between the Donald Danforth Plant Science Center, USA the National Crops Resources Research Institute, Uganda and the Kenya Agricultural Research Institute, Kenya. VIRCA is structured to include all aspects of the intellectual property, technology, regulatory, biosafety, quality control, communication and distribution components required for a GM crop development and delivery process. VIRCA's goal is to improve cassava for resistance to the viral diseases cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) using pathogen-derived RNAi technology, and to field test, obtain regulatory approval for and deliver these products to small landholder farmers. During Phase I of the project, proof of concept was achieved by production and testing of virus resistant plants under greenhouse and confined field trials in East Africa. In VIRCA Phase II, two farmer-preferred varieties will be modified for resistance to CBSD and CMD, and lead events identified after molecular and field screening. In addition to delivery of royalty-free improved planting materials for farmers, VIRCA capacity building activities are enhancing indigenous capability for crop biotechnology in East Africa.