Genomic surveillance uncovers a pandemic clonal lineage of the wheat blast fungus.

Wheat, one of the most important food crops, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America and to guide preemptive wheat breeding for blast resistance.

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.

Smallholder Cassava Planting Material Movement and Grower Behavior in Zambia: Implications for the Management of Cassava Virus Diseases.

Cassava (Manihot esculenta) is an important food crop across sub-Saharan Africa, where production is severely inhibited by two viral diseases, cassava mosaic disease (CMD) and cassava brown streak disease (CBSD), both propagated by a whitefly vector and via human-mediated movement of infected cassava stems. There is limited information on growers' behavior related to movement of planting material, as well as growers' perception and awareness of cassava diseases, despite the importance of these factors for disease control. This study surveyed a total of 96 cassava subsistence growers and their fields across five provinces in Zambia between 2015 and 2017 to address these knowledge gaps. CMD symptoms were observed in 81.6% of the fields, with an average incidence of 52% across the infected fields. No CBSD symptoms were observed. Most growers used planting materials from their own (94%) or nearby (<10 km) fields of family and friends, although several large transactions over longer distances (10 to 350 km) occurred with friends (15 transactions), markets (1), middlemen (5), and nongovernmental organizations (6). Information related to cassava diseases and certified clean (disease-free) seed reached only 48% of growers. The most frequent sources of information related to cassava diseases included nearby friends, family, and neighbors, while extension workers were the most highly preferred source of information. These data provide a benchmark on which to plan management approaches to controlling CMD and CBSD, which should include clean propagation material, increasing growers' awareness of the diseases, and increasing information provided to farmers (specifically disease symptom recognition and disease management options).[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Detection and characterization of fungus (Magnaporthe oryzae pathotype Triticum) causing wheat blast disease on rain-fed grown wheat (Triticum aestivum L.) in Zambia.

Wheat blast caused by Magnaporthe oryzae pathotype Triticum (MoT) is a threat to wheat production especially in the warmer-humid environments. In Zambia, wheat blast symptoms were observed for the first time on wheat (Triticum aestivum L.) grown in experimental plots and five farmers' fields in Mpika district of Muchinga Province during the 2017-18 rainy season. Infected plants showed the typical wheat blast symptoms with the spike becoming partially or completely bleached with the blackening of the rachis in a short span of time. Incidence of blast symptoms on nearly all wheat heads was high and ranged from 50 to 100%. Examination of diseased plant leaves showed the presence of elliptical, grayish to tan necrotic lesions with dark borders on the leaf often mixed with other foliar diseases. A study was conducted to isolate and identify the causal pathogen(s) using classical and molecular methods and determine the pathogenicity of the detected disease causal agent. Morphobiometrical determination of causal pathogen revealed conidia with characteristic pear shaped 2-septate hyaline spores associated with M. oryzae species. Preliminary polymerase chain reaction screening of six isolates obtained from wheat blast infected samples with diagnostic primers (MoT3F/R) was conducted at ZARI, Zambia, and subsequent analysis of two isolates with MoT3F/R and C17F/R was performed at USDA-ARS, USA. Both experiments confirmed that MoT is the causal agent of wheat blast in Zambia. Further, pathogenicity tests performed with pure culture isolates from samples WS4 and WS5 produced typical blast symptoms on all the six inoculated wheat genotypes. Results of this study indicate that MoT is causing wheat blast in rain-fed wheat grown in Zambia, thus making it the first report of MoT in Zambia and Africa. This inter-continental movement of the pathogen (disease) has serious implication for wheat production and trade that needs to be urgently addressed.

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.

Cassava mosaic disease: a review of a threat to cassava production in Zambia.

Cassava (Manihot esculenta Crantz) is one of the most important root staple crops in Zambia. An estimated 30% of Zambians, over 4 million people, consume cassava as part of their daily diet. Cassava is mostly grown by subsistence farmers on fields of less than 1 ha. Cultivation of cassava is hampered by several biotic constraints, of which cassava mosaic disease (CMD) is currently the most important factor limiting cassava production in Zambia. CMD occurs in all the cassava-growing provinces and accounts for 50% to 70% of yield losses countrywide. Strategies to counter CMD were initiated in the early 1990s and included the release of CMD-resistant cassava cultivars. However, efforts to control CMD are limited because few growers plant these cultivars. More recently, to address the CMD problem, regular disease monitoring and diagnostic capabilities have been strengthened, and there is increased support for screening breeders materials. CMD is a rising threat to cassava production in Zambia. This review of CMD research on disease surveillance, CMD spread, yield losses, awareness campaigns and control options in Zambia over the past 25 years informs future control efforts and management strategies.

Cassava Brown Streak Disease and Ugandan cassava brown streak virus Reported for the First Time in Zambia.

A diagnostic survey was conducted in July 2017 in two northern districts of Zambia to investigate presence or absence of cassava brown streak disease (CBSD) and its causal viruses. In total, 29 cassava fields were surveyed and cassava leaf samples were collected from 116 plants (92 symptomatic and 24 nonsymptomatic). CBSD prevalence was approximately 79% (23 of 29) across fields. Mean CBSD incidence varied across fields but averaged 32.3% while mean disease severity was 2.3 on a 1-to-5 rating scale. Reverse-transcription polymerase chain reaction screening of all 116 samples with one generic and two species-specific primer pairs yielded DNA bands of the expected sizes from all symptomatic plants with the generic (785 bp) and Ugandan cassava brown streak virus (UCBSV)-specific (440 bp) primers. All 24 nonsymptomatic samples were negative for UCBSV and all samples tested negative with primers targeting Cassava brown streak virus. The complete genome of a representative isolate of UCBSV (WP282) was determined to be 9,050 nucleotides in length, minus the poly A tail. A comparative analysis of this isolate with global virus isolates revealed its nature as a sequence variant of UCBSV sharing 94 and 96% maximum complete polyprotein nucleotide and amino acid identities, respectively, with isolates from Malawi (MF379362) and Tanzania (FJ039520). This is the first report of CBSD and UCBSV in Zambia, thus expanding the geographical distribution of the disease and its causal virus and further reinforcing the need to strengthen national and regional phytosanitary programs in Africa.

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.