Development of a lateral flow device for in‐field detection and evaluation of PCR‐based diagnostic methods for Xanthomonas campestris pv. musacearum,the causal agent of banana xanthomonas wilt

Xanthomonas campestris pv. musacearum (Xcm) is the causal agent of banana xanthomonas wilt, a major threat to banana production in eastern and central Africa. The pathogen is present in very high levels within infected plants and can be transmitted by a broad range of mechanisms; therefore early specific detection is vital for effective disease management. In this study, a polyclonal antibody (pAb) was developed and deployed in a lateral flow device (LFD) format to allow rapid in‐field detection of Xcm. Published Xcm PCR assays were also independently assessed: only two assays gave specific amplification of Xcm, whilst others cross‐reacted with non‐target Xanthomonas species. Pure cultures of Xcm were used to immunize a rabbit, the IgG antibodies purified from the serum and the resulting polyclonal antibodies tested using ELISA and LFD. Testing against a wide range of bacterial species showed the pAb detected all strains of Xcm, representing isolates from seven countries and the known genetic diversity of Xcm. The pAb also detected the closely related Xanthomonas axonopodis pv. vasculorum (Xav), primarily a sugarcane pathogen. Detection was successful in both naturally and experimentally infected banana plants, and the LFD limit of detection was 10⁵ cells mL^?1. Whilst the pAb is not fully specific for Xcm, Xav has never been found in banana. Therefore the LFD can be used as a first‐line screening tool to detect Xcm in the field. Testing by LFD requires no equipment, can be performed by non‐scientists and is cost‐effective. Therefore this LFD provides a vital tool to aid in the management and control of Xcm.     

The influence of formulation on Trichoderma biological activity and frosty pod rot management in Theobroma cacao

Frosty pod rot (FPR), caused by Moniliophthora roreri, is responsible for significant losses in Theobroma cacao. Due to limited options for FPR management, biological control methods using Trichoderma are being studied. Combinations of three formulations and two Trichoderma isolates were studied between May 2009 and April 2011. The formulations were 0·3 mL L^?1 of the surfactant BreakThru 100SL (BT), a mixture of 1% w/v Sure‐Jell (source of pectin) and 1% w/v potato dextrose broth (PDB) (PP), and an invert oil emulsion of 50% v/v corn oil/2·5% w/v lecithin/0·5% w/v PDB (COP). Water and fungicide, copper oxychloride, were included as controls. Humidity chamber studies indicated that Trichoderma conidia germinated in all formulations if free water was maintained, while only the COP formulation supported germination under drying conditions. In the field, Trichoderma ovalisporum DIS‐70a and Trichoderma harzianum DIS‐219f were applied monthly in each of the three formulations at a rate of 180 mL per tree, 2·46 × 10⁷ conidia per mL. The COP/DIS‐70a formulation provided the largest yield increase compared to all other treatments, including the fungicide control. Averaged over the 2 years, the COP formulation increased yield to 30·7% healthy pods compared to 9·7% healthy pods in the water control. Although the formulation/isolate combinations did not consistently increase endophytic colonization, the PP/DIS‐219f, COP/DIS‐219f and COP/DIS‐70a combinations increased total endophytic/epiphytic colonization by Trichoderma. The invert corn oil formulation of DIS‐70a significantly enhanced yield of healthy cacao pods over 2 years providing a promising model for optimizing Trichoderma‐based biocontrol strategies.     

Foliar resistance of cacao (Theobroma cacao) to Phytophthora palmivora as an indicator of pod resistance in the field: interaction of cacao genotype, leaf age and duration of incubation

The effects were studied of four leaf development stages (LDS) and three durations of incubation (DI) on the accuracy of leaf-disc tests on eight cacao (cocoa) clones (C) for predicting field resistance to phytophthora pod rot caused by Phytophthora palmivora . The clones were known to possess different general combining abilities (GCA) for pod resistance in the field, evaluated monthly at harvest over a 9-year period. Disease severity (DS) was affected strongly by C, DI and LDS, with increasing levels of significance. Two- and three-way interaction effects were smaller than the clone effect, but still significant. Clone effects were most significant for LDS3 (i.e. leaves 50–60 days old) and for DI5 and DI7 (observations made 5 and 7 days after inoculation, respectively). Coefficients of rank correlation between DS and field results were significant for seven of the 12 treatments, with highest values obtained again for treatments LDS3/DI5 ( r = 0·87) and LSD3/DI7 ( r = 0·93). Pooling of data for different LDS and DI treatments did not further improve the correlation with field results. However, these correlations were improved (from an average of 0·74 to 0·88) when the GCA values for field resistance were based on weekly observations, carried out in one year, including losses of pods and cherelles. It was concluded that, when carried out in a standardized manner and under optimal conditions, the leaf-disc test may explain 75–90% of the genetic variation for field resistance of cacao genotypes to P. palmivora .     

Investigations into Encephalartos insect pests and diseases in South Africa and identification of Phytophthora cinnamomi as a pathogen of the Modjadji cycad

South Africa holds the greatest diversity of Encephalartos species globally. In recent years several reports have been received of Encephalartos species in the country dying of unknown causes. The aim of this study was to investigate the presence of, and identify the causal agents of, diseases of Encephalartos species in the Gauteng and Limpopo Provinces of South Africa. Plant material with symptoms and insects were collected from diseased plants in private gardens, commercial nurseries and conservation areas in these regions. Insects collected were identified based on morphology, and microbial isolates based on morphology and DNA sequence data. Insect species identified infesting cultivated cycads included the beetle Amorphocerus talpa, and the scale insects Aonidiella aurantii, Aspidiotus capensis, Chrysomphalus aonidum, Lindingaspis rossi, Pseudaulacaspis cockerelli, Pseudaulacaspis pentagona and Pseudococcus longispinus. Fungal species isolated from diseased plants included species of Diaporthe, Epicoccum, Fusarium, Lasiodiplodia, Neofusicoccum, Peyronellaea, Phoma, Pseudocercospora and Toxicocladosporium. The plant pathogen Phytophthora cinnamomi was identified from E. transvenosus plants in the Modjadji Nature Reserve. Artificial inoculation studies fulfilled Koch's postulates, strongly suggesting that P. cinnamomi is responsible for the deaths of these plants under field conditions.     

Foliar resistance of cacao (Theobroma cacao) to Phytophthora palmivora as an indicator of pod resistance in the field: the effect of light intensity and time of day of leaf collection

Resistance of cacao leaves to Phytophthora palmivora was studied with regard to the time of leaf collection (morning, afternoon) and the degree of exposure of the leaves to light in the field (low, medium and high). The efficiency of leaf disc inoculations in predicting field resistance of nine clones was compared with that of detached and attached pod inoculations. Significant effects were observed, with leaves exposed to high light intensity and collected early in the afternoon showing highest susceptibility. The effect of time of leaf collection was reduced when leaves were stored overnight and leaf discs prepared and inoculated the following day, as compared to inoculations on the day of collection. Interactions between the main factors were significant, though less substantial than the clone effects. The most significant correlations with pod resistance ( r  = 0·70 to 0·97) were obtained for leaves collected early in the morning and exposed to intermediate shade conditions in the canopy. For other treatments, the correlations with pod resistance were still positive ( r  = 0·23 to 0·83) but often not significant. Pod inoculations in the laboratory were better correlated with field resistance ( r  = 0·92) than pod inoculations in the field ( r  = 0·72). Detached pod inoculations were also better correlated with leaf disc inoculations than those of attached pods. The results confirm the validity of laboratory inoculations of leaves and pods to assess field resistance to Phytophthora . Standardization of the leaf disc test is essential to obtain reliable results.     

Genetic analysis of Phytophthora sojae populations in Fujian, China

Phytophthora sojae is a destructive soilborne pathogen causing seedling damping‐off and root rot of soybean (Glycine max). The goal of this study was to determine the genetic structure of P. sojae populations in Fujian, China. Nine microsatellite markers were used to investigate the genetic variation in 19 P. sojae populations, sampled from Fujian Province and northeastern China (Jilin and Heilongjiang Provinces) between 2002 and 2013. Overall, a low genetic diversity, Hardy–Weinberg disequilibrium, and an index (an index of association) that was significantly different from zero were detected in populations; these results were consistent with self‐fertilization and clonal modes of reproduction for this pathogen. However, using Bayesian Markov chain Monte Carlo approach, principal component analysis and neighbour joining (NJ) algorithm, the Fujian P. sojae populations clustered into three distinct groups, one of which included most isolates of the northeast populations. What is more, significant estimates of pairwise fixation indices (F_ST) were detected between most populations, especially in different clusters. It is hypothesized that the cropping system used, the limited dispersal ability, and human‐mediated gene flow may account for the observed genetic structure of P. sojae populations in Fujian, China. In addition, a high virulence frequency of the pathogen on different cultivars carrying known major R genes for resistance, and a rapid increase in virulence frequency, indicated that these major R genes should not be used to manage seedling damping‐off and root rot diseases of soybean (Glycine max).     

Diversity of Avr‐vnt1 and AvrSmira1 effector genes in Polish and Norwegian populations of Phytophthora infestans

The oomycete Phytophthora infestans, the cause of late blight, is one of the most important potato pathogens. During infection, it secretes effector proteins that manipulate host cell function, thus contributing to pathogenicity. This study examines sequence differentiation of two P. infestans effectors from 91 isolates collected in Poland and Norway and five reference isolates. A gene encoding the Avr‐vnt1 effector, recognized by the potato Rpi‐phu1 resistance gene product, is conserved. In contrast, the second effector, AvrSmira1 recognized by Rpi‐Smira1, is highly diverse. Both effectors contain positively selected amino acids. A majority of the polymorphisms and all selected sites are located in the effector C‐terminal region, which is responsible for their function inside host cells. Hence it is concluded that they are associated with a response to diversified target protein or recognition avoidance. Diversification of the AvrSmira1 effector sequences, which existed prior to the large‐scale cultivation of plants containing the Rpi‐Smira1 gene, may reduce the predicted durability of resistance provided by this gene. Although no isolates virulent to plants with the Rpi‐phu1 gene were found, the corresponding Avr‐vnt1 effector has undergone selection, providing evidence for an ongoing ‘arms race’ between the host and pathogen. Both genes remain valuable components for resistance gene pyramiding.     

Relationship between ascochyta blight on field pea (Pisum sativum) and spore release patterns of Didymella pinodes and other causal agents of ascochyta blight

Ascochyta blight of field pea, caused by Didymella pinodes, Phoma medicaginis var. pinodella, Phoma koolunga and Didymella pisi, is controlled through manipulating sowing dates to avoid ascospores of D. pinodes, and by field selection and foliar fungicides. This study investigated the relationship between number of ascospores of D. pinodes at sowing and disease intensity at crop maturity. Field pea stubble infested with ascochyta blight from one site was exposed to ambient conditions at two sites, repeated in 2 years. Three batches of stubble with varying degrees of infection were exposed at one site, repeated in 3 years. Every 2 weeks, stubble samples were retrieved, wetted and placed in a wind tunnel and up to 2500 ascospores g^?1 h^?1 were released. Secondary inoculum, monitored using seedling field peas as trap plants in canopies arising from three sowing dates and external to field pea canopies, was greatest in early sown crops. A model was developed to calculate the effective number of ascospores using predictions from G1 blackspot manager (Salam et al., 2011b; Australasian Plant Pathology, 40 , 621–31), distance from infested stubble (Salam et al., 2011a; Australasian Plant Pathology, 40 , 640–7) and winter rainfall. Maximum disease intensity was predicted based on the calculated number of effective ascospores, soilborne inoculum and spring rainfall over two seasons. Predictions were validated in the third season with data from field trials and commercial crops. A threshold amount of ascospores of D. pinodes, 294 g^?1 stubble h^?1, was identified, above which disease did not increase. Below this threshold there was a linear relationship between ascospore number and maximum disease intensity.     

Molecular and biological characterization of Cowpea mild mottle virus isolates infecting soybean in Brazil and evidence of recombination

The biological and molecular characterization of six isolates of a new Cowpea mild mottle virus strain (CPMMV; Carlavirus, Betaflexiviridae) are reported. Soybean plants with mosaic and stem necrosis were collected in Bahia, Goiás, Mato Grosso and Minas Gerais states, Brazil. Complete genomes of the CPMMV isolates are 8180–8198 nucleotides (nt) long, excluding the 3′‐polyadenylated tail, and have 67–68% nt sequence identity with a Ghana isolate of CPMMV, the only CPMMV isolate for which the genome has previously been sequenced. The replicase has only 60–61% nt sequence identity with the Ghana CPMMV isolate, and the coat protein (CP) is highly conserved (79% nt sequence identity and 95–96% amino acid sequence identity). The high CP identity and the phylogenetic analyses supported the classification of the Brazilian isolates as CPMMV. Biological and molecular differences with the Ghana CPMMV isolate were found and indicated that the six isolates represent a distinct CPMMV strain denominated as CPMMV‐BR. Furthermore, it is shown that recombination occurred mainly in the polymerase gene, and may occur less frequently in other regions of the CPMMV genome.     

Age‐related susceptibility of Eucalyptus species to Phytophthora boodjera

Phytophthora boodjera is a newly described pathogen causing damping off and mortality of Eucalyptus seedlings in Western Australian nurseries. This study evaluated the age‐related susceptibility of several taxa of mallee Eucalyptus to P. boodjera in sterilized washed river sand‐infestation pot trials. Phytophthora cinnamomi and P. arenaria were included for comparison. Seedlings of Eucalyptus taxa were inoculated at 0, 2, 4, 12 and 88 weeks with individual Phytophthora isolates. Pre‐emergent mortality in the presence of Phytophthora was almost 100%. Post‐emergent mortality was 50–100%, depending on isolate, compared to 0% for the control. Mortality was also high for inoculated 1 month‐old seedlings (46–68%) and root length of surviving seedlings was severely reduced. Death from root infection was not observed for seedlings inoculated at 12 and 88 weeks, but they developed root necrosis and reduced root dry weight compared to non‐inoculated controls. Phytophthora boodjera is a pre‐ and post‐emergent pathogen of mallee eucalypts. These eucalypts are susceptible to P. boodjera at all life stages tested, but the mortality rates declined with plant age. Similar results were obtained for P. cinnamomi and P. arenaria. The events leading to its recent appearance in the nurseries remain unknown and further investigations are underway to determine if this is an introduced or endemic pathogen. The approach used here to understand the impact of a Phytophthora species on multiple hosts at different seedling ages is novel and sets a benchmark for future work.     

Phytophthora pachypleura sp. nov., a new species causing root rot of Aucuba japonica and other ornamentals in the United Kingdom

Isolates of an unknown Phytophthora species from the ‘Phytophthora citricola complex’ have been found associated with mortality of Aucuba japonica in the UK. Based on morphological characteristics, growth–temperature relationships, sequences of five DNA regions and pathogenicity assays, the proposed novel species is described as Phytophthora pachypleura. Being homothallic with paragynous antheridia and semipapillate sporangia, P. pachypleura resembles other species in the ‘P. citricola complex’ but can be discriminated by its distinctively thick‐walled oospores with an oospore wall index of 0·71. In the phylogenetic analysis based on three nuclear (ITS, β‐tubulin, EF‐1α) and two mitochondrial (cox1, nadh1) DNA regions, P. pachypleura formed a distinct clade within the ‘P. citricola complex’ with P. citricola s. str., P. citricola E and P. acerina as its closest relatives. Phytophthora pachypleura is more aggressive to A. japonica than P. plurivora and P. multivora and has the potential to affect other ornamental species.     

Evidence for Lettuce big‐vein associated virus as the causal agent of a syndrome of necrotic rings and spots in lettuce

Lettuce big‐vein associated virus (LBVaV, genus Varicosavirus) was shown to be responsible for characteristic necrotic symptoms observed in combination with big‐vein symptoms in lettuce breeding lines when tested for their susceptibility to lettuce big‐vein disease (BVD) using viruliferous Olpidium virulentus spores in a nutrient film technique (NFT) system. Lettuce plants showing BVD are generally infected by two viruses: Mirafiori lettuce big‐vein virus (MiLBVV, genus Ophiovirus) and LBVaV. New mechanical inoculation methods were developed to separate the two viruses from each other and to transfer both viruses to indicator plants and lettuce. After mechanical inoculation onto lettuce plants MiLBVV induced vein‐band chlorosis, which is the characteristic symptom of BVD. LBVaV caused a syndrome of necrotic spots and rings which was also observed earlier in lettuce plants inoculated in the NFT system, resembling symptoms described for lettuce ring necrosis disease (RND). This observation is in contrast with the idea that LBVaV only causes latent infections in lettuce. De novo next‐generation sequencing demonstrated that LBVaV was the only pathogen present in a mechanically inoculated lettuce plant with symptoms, providing evidence that LBVaV was the causal agent of the observed necrotic syndrome and thus fulfilling Koch’s postulates for this virus. The necrotic syndrome caused by LBVaV in lettuce is referred to as LBVaV‐associated necrosis (LAN).     

Virulence of ‘Dickeya solani’ and Dickeya dianthicola biovar‐1 and ‐7 strains on potato (Solanum tuberosum)

Studies were conducted to explain the relative success of ‘Dickeya solani’, a genetic clade of Dickeya biovar 3 and a blackleg‐causing organism that, after recent introduction, has spread rapidly in seed potato production in Europe to the extent that it is now more frequently detected than D. dianthicola. In vitro experiments showed that both species were motile, had comparable siderophore production and pectinolytic activity, and that there was no antagonism between them when growing. Both ‘D. solani’ and biovar 1 and biovar 7 of D. dianthicola rotted tuber tissue when inoculated at a low density of 10³ CFU mL^?1. In an agar overlay assay, D. dianthicola was susceptible to 80% of saprophytic bacteria isolated from tuber extracts, whereas ‘D. solani’ was susceptible to only 31%, suggesting that ‘D. solani’ could be a stronger competitor in the potato ecosystem. In greenhouse experiments at high temperatures (28°C), roots were more rapidly colonized by ‘D. solani’ than by biovar 1 or 7 of D. dianthicola and at 30 days after inoculation higher densities of ‘D. solani’ were found in stolons and progeny tubers. In co‐inoculated plants, fluorescent protein (GFP or DsRed)‐tagged ‘D. solani’ outcompeted D. dianthicola in plants grown from vacuum‐infiltrated tubers. In 3 years of field studies in the Netherlands with D. dianthicola and ‘D. solani’, disease incidence varied greatly annually and with strain. In summary, ‘D. solani’ possesses features which allow more efficient plant colonization than D. dianthicola at high temperatures. In temperate climates, however, tuber infections with ‘D. solani’ will not necessarily result in a higher disease incidence than infections with D. dianthicola, but latent seed infection could be more prevalent.     

Survey of viruses infecting open‐field pepper crops in Côte d'Ivoire and diversity of Pepper veinal mottle virus and Cucumber mosaic virus

The prevalence of viruses in pepper crops grown in open fields in the different agro‐ecological zones (AEZs) of Côte d'Ivoire was surveyed. Pepper veinal mottle virus (PVMV; genus Potyvirus) and Cucumber mosaic virus (CMV; genus Cucumovirus) were the most frequent viruses among those surveyed, while tobamoviruses (genus Tobamovirus) were detected at low frequency. PVMV showed a high heterogeneity across AEZs, which may be related to climatic, ecological or agronomical conditions, whereas CMV was more homogeneously distributed. The molecular diversity of CMV and PVMV were analysed from partial genome sequences. Despite the low number of CMV isolates characterized, two molecular groups were revealed, one corresponding to subgroup IA and the other to reassortants between subgroups IA and IB. RNAs 1 and 3 of the reassortants clustered with the IB subgroup of CMV isolates, whereas their RNA 2 clustered with the IA subgroup. Importantly, RNA 1 of CMV isolates of the IB subgroup has been shown to be responsible for adaptation to pepper resistance. The diversity of PVMV in the VPg‐ and coat protein‐coding regions revealed multiple clades. The central part of the VPg showed a high level of amino acid diversity and evidence of positive selection, which may be a signature of adaptation to plant recessive resistance. As a consequence, for efficient deployment of resistant pepper cultivars, it would be desirable to examine the occurrence of virulent isolates in the CMV or PVMV populations in Côte d'Ivoire and to follow their evolution as the resistance becomes more widely deployed.     

Metabarcoding and development of new real‐time specific assays reveal Phytophthora species diversity in holm oak forests in eastern Spain

The evergreen holm oaks (Quercus ilex subsp. ilex and Q. ilex subsp. ballota) are the most representative tree species in the Iberian peninsula and the main tree species in oak‐rangeland ecosystems (dehesas). Oak decline in western, central and southern parts of Spain has been associated with root rot caused by Phytophthora cinnamomi for decades. However, Phytophthora species such as P.  quercina and P. psychrophila have recently been found associated with Quercus decline in eastern Spain where calcareous soils are predominant. Soil and root samples from two Quercus forests presenting decline symptoms in two different geographical areas in eastern Spain (Carrascar de la Font Roja and Vallivana) were analysed by amplicon pyrosequencing. Metabarcoding analysis showed Phytophthora species diversity, and revealed that an uncultured Phytophthora taxon, named provisionally Phytophthora taxon ballota, was the predominant species in both areas. In addition, a real‐time PCR assay, based on the pyrosequencing results, was developed for the detection of this uncultured Phytophthora taxon, and also for the detection of P. quercina. TaqMan assays were tested on soil and root samples, and on Phytophthora pure cultures. The new assays showed high specificity and were consistent with metabarcoding results. A new real‐time PCR protocol is proposed to evaluate the implication of different Phytophthora spp. in oak decline in eastern Spain.     

Analysis of resistance to witches’ broom disease (Moniliophthora perniciosa) in flower cushions of Theobroma cacao in a segregating population

This study evaluates resistance to witches’ broom disease in flower cushions of Theobroma cacao under field conditions. The aim was to determine optimal inoculation methods to evaluate the disease incidence using flower cushions in the field. A segregating mapping population of 580 trees (cultivar TSH 1188 × CCN 51) was analysed under two field conditions: high and low inoculum levels (in different years), corresponding respectively to trees with or without dried witches’ brooms hanging on the trees and producing basidiocarps. The number of newly formed cushion brooms in each tree was counted by the conventional method, and also the healthy and infected flower cushions in three 30 cm‐long regions along the trunk and the two main branches. The field inoculation methods discriminated between genotypes, with a 26% increase in disease incidence by Moniliophthora perniciosa at high inoculum. Two different segregation patterns were also observed: 27:27:9:1 under low, and 27:9:9:9:3:3:3:1 under high inoculum potential. It was also determined that at least 20 flower cushions were needed to accurately determine the percentage of infection. These methodologies allowed identification of the extreme phenotypes in this mapping population, and can therefore facilitate the detection of sources of resistance to witches’ broom disease.     

Evidence for seed transmission and symptomless growth of Ramularia collo‐cygni in barley (Hordeum vulgare)

Ramularia collo‐cygni (Rcc) is becoming an increasing problem for barley growers across Europe. However, the life cycle of the pathogen is only slowly being elucidated. In this study, Rcc DNA was detected in a number of harvested seed samples from 1999 to 2010, with mean levels peaking in winter barley samples in 2009. A number of experiments were carried out to determine whether the pathogen could move from barley seed to seedlings, and also from seed through the developing plant and into the subsequent generation of seed, both in controlled experiments and in field trials. Results from testing of seed indicated that the fungus is widespread at the end of the growing season in harvested grain samples and can be transmitted to developing plants from infected seed stock. Examination of infected seedlings did not reveal the presence of spores but fungal structures were found within the leaf. The location of the fungus within seed was examined, with Rcc DNA found in both embryo and non‐embryo tissue. The implications for barley production of the pathogen being seedborne are discussed.     

Distribution and genetic diversity of root‐knot nematodes (Meloidogyne spp.) in potatoes from South Africa

A molecular‐based assay was employed to analyse and accurately identify various root‐knot nematodes (Meloidogyne spp.) parasitizing potatoes (Solanum tuberosum) in South Africa. Using the intergenic region (IGS) and the 28S D2–D3 expansion segments within the ribosomal DNA (rDNA), together with the region between the cytochrome oxidase subunit II (COII) and the 16S rRNA gene of the mtDNA, 78 composite potato tubers collected from seven major potato growing provinces were analysed and all Meloidogyne species present were identified. During this study, M. incognita, M. arenaria, M. javanica, M. hapla, M. chitwoodi and M. enterolobii were identified. The three tropical species M. javanica, M. incognita and M. arenaria were identified as the most prevalent species, occurring in almost every region sampled. Meloidogyne hapla and M. enterolobii occurred in Mpumalanga and KwaZulu‐Natal, respectively, while M. chitwoodi was isolated from two growers located within the Free State. Results presented here form part of the first comprehensive surveillance study of root‐knot nematodes to be carried out on potatoes in South Africa using a molecular‐based approach. The three genes were able to distinguish various Meloidogyne populations from one another, providing a reliable and robust method for future use in diagnostics within the potato industry for these phytoparasites.     

High throughput real‐time RT‐PCR assays for specific detection of cassava brown streak disease causal viruses,and their application to testing of planting material

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is causing severe losses in cassava production in Kenya, Tanzania and Uganda. Two real‐time RT‐PCR assays based on TaqMan chemistry capable of detecting and distinguishing these two viruses are described. These assays were used to screen 493 cassava samples collected from western and coastal Kenya, the main cassava regions of Uganda and inland Tanzania. Both viruses were found in all three countries and across regions therein. Association of CBSD leaf symptom status with CBSV and UCBSV assay results was weak, confirming the need for a diagnostic assay. For leaf samples that were observed with CBSD‐like leaf symptoms but shown as CBSV and UCBSV negative by the RT‐PCR assay, deep sequencing using a Roche 454 GS‐FLX was used to provide additional evidence for the absence of the viruses. The probability of the CBSD associated diagnostics detecting a single CBSV or UCBSV positive sample amongst other non‐CBSD samples was modelled. The results of this study are discussed in the context of the application of diagnostics of CBSD‐associated viruses under the Great Lakes Cassava Initiative and the need to minimize the risk of further spread of the viruses with cassava multiplication material. It is shown that high throughput testing undertaken at Fera of 300 cassava leaves taken from fields for seed multiplication, when analysed in pools of 10, has given a 95% probability of detecting 1% infected plants in the field.