Farmer-Cultivated Landraces and Improved Cassava Genotypes Exhibit Varied Response to Cassava Brown Streak Disease Under Natural Infections in Kenya

Cassava brown streak disease (CBSD) affects cassava growth and production with resultant foliar symptoms and storage root necrosis causing significant economic losses to farmers. The disease is caused by either Cassava brown streak virus (CBSV) or Uganda cassava brown streak virus (UCBSV), both vectored by whitefly, Bemisia tabaci . Cultivation of CBSD-free or resistant cassava genotypes offers the most long term sustainable CBSD management. Through a field survey, the present study assessed variations for CBSD infections amongst local cassava landraces (LCLs) and improved genotypes (IPGs) cultivated by farmers in Kenya. Foliar symptoms were scored through CBSD severity (CBSD-S) and CBSD incidence (CBSD-I) and relative CBSV and UCBSV titres measured through quantitative RT-PCR with COX as reference gene. Overall results showed varied mean CBSD-I of 12-52% and CBSD-S of 2.0-3.8 between surveyed regions while CBSD-I of 0-100% and CBSD-S of 1.0-4.6 (asymptomatic - severe) between cassava germplasm. The symptoms varied significantly (P ≤ 0.001) enabling identification of CBSD tolerant germplasms (CTGs). RT-PCR revealed 35% no infection, 49% single infections and 16% dual infections. CBSV infected more germplasm (64%) and exhibited higher viral titres compared to UCBSV at 16%. Correlation between CBSD-S and CBSV titre was positive (r = 0.78) and significant (P ≤ 0.004) implicating CBSV with CBSD symptom expression. Out of 112 assessed germplasm, 56% showed low CBSD-S scores and low viral titre thus categorized as CTGs. About 71% of IPGs were CTGs compared to 47% of LCLs. This implied that 29% IPGs and 53% LCLs sampled in this study were CBSD-infected. This indicated potential breakdown of CBSD resistance overtime especially in IPGs, hence could hamper CBSD management. However, the CBSD-free IPGs and LCLs identified in this study could also be bulked for distribution to cassava farmers as clean planting materials for improved cassava production. They could also be potential genetic resources for introgression of virus-resistance traits into farmer-preferred


Introduction
Cassava (Manihot esculenta Crantz) is a perennial woody shrub in the Euphorbiaceae family native to South America but now grown in tropical and subtropical areas worldwide for its edible starchy tuberous roots [1,2]. Majority of cassava cultivation occurs in Africa, where it is gaining popularity due to its low water requirement, survivability in marginal soils, yield under adverse conditions and flexibility in harvest time [3,4]. Due to its inherent tolerance to drought stress [5], the crop has become an important staple for food-vulnerable populations in sub-Saharan Africa (SSA) [6,7]. Despite this significance, cassava's strategic importance as food security crop in SSA [8] has been rendered vulnerable by major viral diseases such as cassava brown streak disease (CBSD) which persistently reduces overall storage root quality and quantity resulting in up to 100% yield losses [9]. CBSD has caused approximately US$750 million annual losses in Kenya, Tanzania, Uganda and Malawi [10]. CBSD is caused by two species of single-stranded RNA viruses (+ssRNA), Cassava brown streak virus (CBSV) and Uganda cassava brown

Field survey
Through a field survey carried out in April 2018, a total of 112 cassava germplasm (Table 1) were assessed for CBSD severity (CBSD-S) and CBSD incidence (CBSD-I) in five major cassava growing areas of Kenya ( Figure 1). The multi-stage field survey design and systematic identification of farms for sample collection was adopted from Koima, et al. [24] and Mware, et al. [15]. This involved selection of cassava plants more than three months old at regular intervals of 2 to 5 km along motorable roads traversing each sampling location. In each farm, 30 plants were sampled at regular intervals along the diagonals of an X-shaped transect (IITA, 2009;27]. Farmers providing the names and age of the cassava variety or genotype on their farms (Table 1). CBSD-S was visually scored on a scale of 1-5 as described by Abaca, et al. [25] where 1 = no apparent symptoms; 2 = slight foliar chlorotic leaf mottle, no stem lesions; 3 = foliar chlorotic mottle and blotches with mild stem lesions, no die-back; 4 = foliar chlorotic mottle and blotches and pronounced stem lesions with no die-back severe and 5 = defoliation with stem lesions and pronounced die-back. CBSD-I was determined as the proportion number of CBSD symptomatic plants relative to total number of number of plants assessed. of whitefly vectors and associated changing environmental and climatic conditions [20,21].
Foliar CBSD symptoms include feathery chlorosis along the veins and chlorotic mottling of old leaves, brown elongate necrotic lesions on young green stems with die-back from the shoot tip and brown, corky, necrotic tuberous storage roots with characteristic constrictions [20,22]. The affected roots are unusable as source of food and valueless in the marketplace [20] thus threatening the food and economic security of East and Central Africa [23]. Thus, the impact of CBSD has important implications for smallholder cassava farmers and rural communities [20]. Restriction (quarantine) of movement of planting materials from regions with reported CBSD incidences, control of whiteflies and breeding or development and deployment of CBSD-tolerant/resistant cassava germplasm are some mitigation measures that have been recommended to reduce CBSD-associated yield losses in farmer fields. To contribute towards management of CBSD, the present diagnostic study assessed variations in CBSD infections in local cassava landraces (LCLs) and improved cassava genotypes (IPGs) sampled from farmer fields in Kenya. The objective was to compare response to CBSD between IPGs and LCLs under natural infection by CBSVs. This could aid in identification and selection of CBSD-free or resistant germplasm for bulking and production of clean planting materials and for CBSD resistance breeding purposes. manufacturer's recommendations (Thermo Fisher Scientific, Waltham, MA, USA). DNase-treated RNA were then subjected to cDNA synthesis using AccuPower® RocketScript TM Cycle RT First strand cDNA Synthesis kit following manufacturer (Bioneer, Daejeon, South Korea) instructions. Due to differences in RNA quantity, the samples were normalized to a working concentration of 100 ng/µl by addition of an appropriate amount of sterile water [27]. Duplex reverse transcriptase polymerase chain reaction (RT-PCR) was performed on the synthesized complementary DNA (cDNAs) for simultaneous detection of both CBSV and UCBSV in a single reaction. Primers CBSVF2 (5'-GGRCCATACATYAARTGGTT-3'), CBSVR7 (5'-CCCTTTGCAAARCTRAAATARC-3') and CBSVR7 (5'-CCATTRTCTYTCCAMADCTTC-3') were used to amplify approximately 441 and 345 bp of UCBSV and CBSV respectively

Virus diagnostics and quantification
Out of the 30 pre-selected plants per cassava germplasm, five plants per germplasm (biological replicates) were randomly sampled for CBSV and UCBSV diagnostics and quantification. Both fourth and fifth fully expanded leaf from the top of each plant [26] was harvested and pooled into one sample. Total RNA was extracted from three biological replicates per germplasm using CTAB-based protocol as described by Orek [65]. Quantity, quality and integrity of each RNA sample was measured using NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 1.5% agarose gel electrophoresis (AGE). All RNA isolates were digested by RNAse-free DNase I to eliminate genomic cassava DNA contamination according Using 7900 Fast RT PCR System (Applied Biosystems®, Foster City, CA), real-time quantitative PCR (RT-qPCR) was performed on the synthesized cDNA samples to determine the virus loads. The RT-qPCR was based on TaqMan chemistry with primers and probe sequences adopted from Adams, et al. [29]. CBSV probe was 5'-FAM-TAMRA-3' labeled, UCBSV probe 5'-VIC-TAMRA-3' labeled and cytochrome oxidase gene (COX) used as an internal control with primers COX-F (5'-CGTCGCATTCCAGATTATCCA-3'), COX-R (5'-CAACTACGGATATATAAGRRCCRRAACTG-3') and probe (5'-[FAM]-AGGGCATTCCATCCAGCGTAAGCA-[TAMRA]-3') [29]. Mitochondrial COX is a widely used reference or endogenous gene to normalize cycle threshold (Ct) values validated by Adams, et al. [29] and has been widely used for CBSV and UCBSV quantification [27,30,31]. The reaction composed of three cDNA samples (biological replicates) for each cassava germplasm and three technical replicates per biological replicate. The 20 µl reaction volume consisted of 10 µl DreamTaq PCR master mix (2X) (Thermo Fisher Scientific, Waltham, MA, USA), 1 µl each forward and reverse primer (7.5 µM), 0.5 µl Taqman probe (5 µM), 2 µl cDNA template and 5.5 µl nuclease-free sterile H 2 O. For every assay, nontemplate H 2 O control (no cDNA) was included to check for reagent contamination. The reactions were incubated at 95°C for 10 min for initial denaturation, followed by 40 cycles at 95°C for 15 sec, annealing at 60°C for 1 min and extension at 60°C for 1 min (Ct collected).

Data Analysis
Descriptive statistics was applied in analysis of CBSD-S and CBSD-I using SPSS software version 22.0 with group means separated based on standard deviations to determine significant differences amongst germplasm. For RT-PCR, presence (+) or absence (-) of CBSV (345 bp) and UCBSV (441 bp) was based on the detectability of the expected RT-PCR band sizes as visualized underage. For RT-qPCR, Ct values collected at 60°C for 1 min, were generated on default settings of the 7900 Fast System SDS software and then used to compute CBSV and UCBSV fold change (virus loads or titres) in a cassava germplasm relative to COX reference gene using the comparative 2 -∆∆CT method [32]. All cassava germplasm that had Ct values of 40 for UCBSV or CBSV were considered to be free of these viruses [27]. Variations for mean CBSD-S and viral fold change (VFC) or load were used to classify each germplasm as either CBSD tolerant germplasm (CTGs) or CBSD susceptible germplasm (CSGs). CTGs had mean foliar CBSD-S scores of 0.0-2.4 (no apparent symptoms to slight foliar chlorotic leaf mottle) with low VFC (0.0-1.4), while CSGs had foliar CBSD-S scores of 2.5-5.0 (foliar chlorotic mottle to defoliation with stem lesions and pronounced dieback) and high VFC (> 1.5). Pearson correlation coefficient (r) was used to determine relationships between virus load or VFC, CBSD-S and CBSD-I.

RT-PCR diagnosis of CBSV and UCBSV
Detection of CBSV and UCBSV through the duplex RT-PCR revealed no infection, single infections (CBSV or UCBSV)      Table 4). The different levels of infections were shown by the 441 bp and 345 bp amplicons sizes of UCBSV and CBSV respectively ( Figure 5). Generally, out of 112 tested germplasm, 35% were non-infected, 49% had single infection and 16% had dual infection (Figure 6a). For specific germplasm, majority (57%) of IPGs compared to 23% LCLs were non-infected, most (60%) of LCLs had single infections compared to 31% IPGs and dual infections were respectively analyzed in 12 and 17% of IPGs and LCLs (Figure 6a). For specific viruses, generally CBSV and UCBSV were respectively detected in 64 and 14% of combined cassava germplasm (Figure 6b). For different germplasm, CBSV was respectively detected in 43 and 77% of IPGs and LCLs compared to UCBSV that infected in 12% IPGs and 16% LCLs (Figure 6b).

Correlations between virus load and CBSD symptom
All the 2-tailed correlation coefficients (r) between CBSD symptoms and virus load were strong positive and significant ( Table 6), (Figure 8). For example, r = 0.78 and P ≤ 0.004 for CBSD severity with CBSV load (Table 6a) and r = 0.51, P ≤ 0.006 for CBSD incidence and CBSV load (Table 6b). Weak and positive correlations were observed between UCBSV loads, severity and incidence. For example, r = 0.25, P ≤ 0.008 for CBSD severity and UCBSV load (Table 6c) and r = 0.24, P ≤ 0.01 for CBSD incidence and UCBSV load (Table 6d). Correlations between CBSV load and UCBSV load was also positive and significant (r = 0.43, P ≤ 0.03) ( Table 6e). Correlations of determination (r 2 ) was also calculated and graphed between log10 of the virus titre fold change and mean CBSD severity ( Figure 8). The correlations results showed weak association with r 2 = 0.15 for severity with CBSV titre (Figure 8a) and r 2 = 0.04 for severity and UCBSV titre (Figure 8b).

Discussion
The present survey identified asymptomatic and symptomatic cassava plants with the latter exhibiting typical CBSD symptoms such as leaf chlorosis that appeared yellowish and mottled and yellowish to dark-brown constricted and necrotic storage roots. Previous studies have characterized similar CBSD symptoms in cassava [9,20,27,33,34]. The necrotic lesions on the storage roots render them inedible and unmarketable resulting in economic losses for farmers. The significant variations for foliar CBSD severity and incidence between surveyed regions and amongst cassava germplasm reported in this study could have three implications. First it can enable identification of CBSD hot spots across the country that can be used establish resistance breeding programs. In contrast, such hot spots could also be recommended for quarantine measures to restrict the spread of CBSD. Secondly, the wider genotypic variations for CBSD can aid in selection of CBSD-free planting materials or parental sources for CBSD resistance breeding. Lastly, regions with low or no CBSD could be used to establish and bulk CBSD-free seeds or cuttings for distribution to farmers in affected areas. Planting CBSD-free cuttings is a mitigation measure that reduces the effects of CBSD on cassava yield [35][36][37]. The present study identified Siaya (0°N; 33°E) and Kakamega (0°N; 34°E) in western Kenya as CBSD hot spots and Kitui (0°S; 37°E) in eastern as an   area with low CBSD infections. Further, the study identified about 56% CBSD resistant and 44% CBSD susceptible cassava germplasms. These regional and cassava germplasm variations for CBSD severity and incidence were corroborated by relatively similar reports of related studies in Kenya [15,24,38,39]. The reasons that have been attributed to these variations include occurrence, distribution, and abundance of the whitefly vector (Bemisia tabaci) [40,41], virus species (CBSV or UCBSV) causing the symptoms [42,43], differential cultivar sensitivity to CBSD (15,38], age or growth stage of the plant [9,44,45], dissemination of infected planting materials [19,40] and varied growing or weather conditions [42,46].

Germplasm CBSV UCBSV LVI Germplasm CBSV UCBSV LVI Germplasm CBSV UCBSV LVI
Molecular detection and quantification of CBSVs can identify virus-latent genotypes and explain observed variations in symptom severity. In this study, the presence/ absence, and concentrations of CBSV and UCBSV in cassava germplasm were respectively determined by duplex RT-PCR and RT-qPCR. This resulted in identification of CBSVs-free germplasms as well those with single and dual or mixed infections of CBSV and UCBSV. Results showed single infections with high CBSV concentration as predominant and UCBSV exhibiting minor infections with low titres. Similarly, Mukiibi, et al. [30] reported more prevalent single-CBSV infections compared to single-UCBSV infections. Although dual or co-infections with both virus species is common [14,47], only 16% of all germplasm had dual infections in the present study. Genotypic differences for CBSV and UCBSV were also observed where most improved genotypes were CBSVs-free or with low viral loads compared to local landraces that had high concentrations of CBSV. This concurs with previous research which similarly reported higher concentration of CBSV and insignificant or low UCBSV in cassava varieties [27,48].
The genotypic variations in response to both viruses could perhaps be associated with differences in mechanism of host-virus interactions between CBSV and UCBSV [46]. The large differences in virus loads could also be due to competition between the viruses with CBSV outcompeting UCBSV, differences in pathogenicity or differential reaction of genotype to each virus [27]. Compared to UCBSV, CBSV is a more aggressive and virulent CBSD viral pathogen generally accumulating higher titres in most host genotypes in either field or controlled experiments [13,14,27,42,49,50]. Further, CBSV isolates are often more detectable, have higher infection rates and induces more severe symptoms in cassava compared to the "less virulent" UCBSV that generates milder foliar symptoms [42,48]. Although both CBSV and UCBSV cause similar symptoms, the underlying factors behind their varied virulence levels are yet to be established to date [13,42,49,50]. In summary, response CBSV and UCBSV is genotype specific.
Possible inferences could be drawn from the significantly positive correlations observed between virus loads and foliar severity or incidences. For instance, higher CBSD severity scores could be associated with increased accumulation or concentration of CBSVs in a plant. Indeed, in the present study, improved genotypes and landraces that had higher visual CBSD severity also showed higher concentration of viruses especially CBSV. This however contrasted with other findings that did not correlate symptom severity with viral load [27,61]. Ogwok, et al. [50] similarly reported no clear correlation between CBSVs and CBSD symptom types and variability. Although positive, the weak correlations observed between CBSD severity and UCBSV load, implied the lesser or limited effect of UCBSV on CBSD symptoms in the present study. This further confirmed UCBSV as a "lesser" virulent species. Shirima, et al. [62] also reported insignificant or weak correlation between UCBSV titre and symptom severity. The significant and positive correlation between CBSV load and UCBSV load implied potential synergistic effect or interaction  between the two viruses. Higher CBSV titres has been recorded in cassava varieties with dual or mixed infections than in singly infected samples suggesting the possible existence of synergism between CBSV and UCBSV in field-infected cassava [63]. CBSD symptoms depend on whether cassava is infected with a single virus, or if there is a concurrent infection of two or more viruses resulting in synergistic interactions [64].
In the current study, 16% of the germplasm that had dual infections also had higher CBSD foliar severity or incidences and viral loads.

Conclusion
In conclusion, the present survey revealed differences in response to CBSD infections between local cassava landraces and improved genotypes with more candidates from the later exhibiting lesser CBSD severity or incidences and viral loads. Most improved genotypes were therefore considered tolerant or resistant to CBSD. However, some of the improved genotypes were also susceptible to CBSD implying potential breakdown of CBSD resistance overtime. Although most local landraces were susceptible to CBSD, a few of them were CBSD-free. Both CBSD-free or resistant germplasms (improved genotypes and landraces) could be bulked for distribution to cassava farmers as clean planting materials for improved production. They could also be potential genetic resources to introgress virus-resistance traits into farmerpreferred varieties.