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Quinoa (Chenopodium quinoa Willd.) is a native American crop mainly grown in the Andes of Bolivia and Peru. During the last decades, the cultivation of quinoa has expanded to more than 125 countries. Since then, several diseases of quinoa have been characterized. A leaf disease was observed on quinoa plants growing in an experimental plot in Eastern Denmark in 2018. The symptoms produced by the associated fungi consisted of small yellow blotches on the upper surface of leaves with a pale chlorotic halo surrounding the lesion. These studies used a combination of morphology, molecular diagnostics, and pathogenicity test to identify two different Alternaria species belonging to Alternaria section Infectoriae and alternata as the causal agent of observed disease symptoms. To the best of our knowledge, this is the first report of Alternaria spp. as foliar pathogens of quinoa. Our findings indicate the need for additional studies to determine potential risks to quinoa production.

Cassava is Africa’s most important food security crop and sustains about 700 million people globally. Survey interviews of 320 farmers in three regions of Tanzania to identify their production characteristics, and interviews with 20 international whitefly/virus experts were conductedto identify adaptation strategies to lessen the impacts of cassava whiteflies and viruses due to climate change in Tanzania. Structured and pre-tested interview schedules were conducted using a multistage sampling technique. Most of the farmers (66.8%) produced cassava primarily for food, and relied mainly on their friends (43.8%) and their farms (41.9%) for cassava planting materials. Farmers significantly differed in their socio-economic and production characteristics except for gender and access to extension support (P < 0.01). A significant association was found between extension support, sources of planting materials, and reasons for growing cassava with both the control of cassava viruses and the control of whiteflies by the farmers. A significantly higher number of farmers controlled cassava viruses (38.1%) than cassava whiteflies (19.7%). The adaptation strategies most recommended by experts were: integrating pest and disease management programs, phytosanitation, and applying novel vector management techniques.The experts also recommended capacity building through the training of stakeholders, establishing monitoring networks to get updates on cassava pests and disease statuses, incorporating pest and disease adaptation planning into the general agricultural management plans, and developing climate change-pest/disease models for accessing the local and national level impacts that can facilitate more specific adaptation planning in order to enhance the farmers’ adaptive capacities.

The diversity and genetic differentiation of populations of Fusarium species associated with sorghum fields, both endophytes obtained from sorghum performing and non performing plants and isolates obtained from two sampling periods were investigated. Fusarium specific Internal Transcribed Spacer 2 (FITS2) primers set were used to assess genetic variability of 32 isolates from susceptible Fusarium spp. endophytes from Sorghum tissues. Fusarium thapsinum (Gibberella thapsina) with 68.75% of the isolates constituted the majority of Fusarium spp. isolated in performing plants. Gibberella thapsina species identified are described as non-pathogenic and associated to performing plant of sorghum. Previously, some species of Fusarium thapsinum have been recognized as pathogenic and responsible for yield losses in several cereal crops including Sorghum bicolor produced in Burkina Faso. The other Fusarium spp. identified in this study including Fusarium subglutinans, Fusarium chlamydosporum, Gibberella intermedia, Fusarium dlaminii, Fusarium oxysporum, Fusarium proliferatum, and Fusarium spp. An additional unknown fungi species were also identified. A diverse population of 10 sequence types was found, although 8 sequence types represented nearly two-thirds of the isolates studied. The sequence types were placed in different phylogenetic clades within Fusarium spp., and endophytic isolates were not monophyletic. Phylogenetic analysis from Neighbor-Joining/UnWeighted Neighbor-Joining showed a high genetic relationship among these 32 isolates of Fusarium spp. and high variation in FITS sequence of them. The use of specific phylomarker of the genus Fusarium allowed to identify the endophytic species of this genus and to establish the phylogenetic relationships between the endophytic species of Fusarium. The phylogenetic analysis revealed three groups of the fungi. However, no relationship between these groups and the geographical origins of these fungi has been established. Key words: Fusarium thapsinum, endophyte, FITS2 marker, sorghum.

Crops with deeper rooting is an emerging tool for better exploitation of soil resources. However, there is a need for more in-depth understanding on how the increased rooting depth may be achieved. In this study a novel approach for obtaining deeper rooting has been proposed. Crops with assumed similar capacity for subsoil exploration: sugar beet (Beta vulgaris) and chicory (Cichorium intybus var. foliosum) were intercropped. Repeated measurements of biomass, deep root growth, and nutrient uptake were conducted to monitor plant competitive dynamics in the intercrop and sole crops. It was found that the intercrop positively affected biomass production with Land Equivalent Ratio close to or greater than 1 (0.99 – 1.14). Similarly, the strongest root growth over time was observed for the intercrop (from 98 ± 48 to 304 ± 28 cm depth). Moreover, the effect from the interspecific interactions in the intercrop varied over time. In the first half of the season yield advantage and the observed enhanced contribution to the uptake of N, Mg, Mn, Zn, and Na in the intercrop were driven by the sugar beet. Later in the growing season, yield advantage, deep root growth, and contribution to the uptake of S, Fe, Cu, and Al in the intercrop were driven by the chicory. This has also been confirmed by the root quantification analysis, which showed that in the end of the season intercrop consisted of 84 % and 98 % roots from the chicory at 1 and 2.5 m depth, respectively. This study concluded that intercropping two crops with similar root characteristics, sugar beet and chicory, can still lead to complementary interactions showing potential for efficient deep soil exploration by roots and yield advantage in comparison with the sole crops.

The whiteflies belonging to the species group Bemisia tabaci transmit viruses that are the greatest constraint to the production of cassava, Africa’s most important food security crop. We studied the life history of an endemic African population of B. tabaci SSA-ESA at six constant temperatures (16, 20, 24, 28, 32, 36 °C) in controlled environment chambers, and for 10 generations in the field. Oviposition occurred between 20 and 28 °C, with highest fecundity (114.5 ± 88.2) eggs per female at 20 °C. Similarly, B. tabaci adult females average survival time was longest (19.7 days) at 24 °C, and shortest (8.5 days) at 36 °C. Immature development time decreased with increasing temperature from 59.3 days at 16 °C to 16.3 days at 28 °C, and increased above 28 °C. A constant temperature of 36 °C was lethal to eggs and first instars and no immature stage developed further to adults at this temperature. Several temperature-driven models were fitted for describing the species development, survival and reproduction using Insect Life Cycle Modelling (ILCYM®) software. Median temperature-dependent development rates in individual immature life stages were statistically described by Taylor, Briere 1 and Tb (Logan) models. Temperature-dependent mortality and senescence rates in immature stages were described by using parabolic nonlinear models. The longevity of adult females and oviposition time was best described by the Weibull distribution. Maximum population growth was predicted between 26 and 28 °C. These results will be useful for spatio-temporal analysis of climate change impacts on the distribution and abundance of this pest.

The diversity of fungal endophytes in Sorghum bicolor was investigated in samples collected from 10 different geographical regions of Karnataka state, India. A total of 360 endophytes were isolated from leaf, stem, and root tissues and were assigned to 26 fungal species based on morphology and molecular characterization using ITS sequences. All the endophytes belonged to the phylum Ascomycota. The diversity (Shannon H, 2.57; Simpson_1‐D, 0.92) and species richness (Margalef's, 4.68; Menhinick, 3.61) were found to be higher for the endophytes isolated from leaf tissues. The species evenness of the endophytic assemblage was strongly influenced by tissue type, followed by geographical location. The biocontrol potential of isolated endophytes was evaluated against economically destructive sorghum grain mould pathogens such as Fusarium thapsinum, Epicoccum sorghinum, Alternaria alternata, and Curvularia lunata using the dual culture method. Biocontrol potential was exhibited by 26 endophytic isolates, of which Trichoderma asperellum recorded broad‐spectrum activity against target pathogens, followed by E. nigrum and A. longipes. Most (82%) endophytes showed plant growth‐promoting traits. Biosynthesis of indole‐3‐acetic acid (IAA) was observed in 84% of isolates, and phosphate solubilization, siderophore production, and cellulase activity was observed in 69%, 23%, and 27% of isolates, respectively. Seeds treated with T. asperellum exhibited a significantly higher seed vigour index (2096), germination percentage (94%), and yield under greenhouse and field conditions. The results were substantiated by the confocal microscopy analysis, which clearly demonstrated the colonization of treated endophyte in root tissues. The present study reveals an ecofriendly approach to explore T. asperellum in sorghum disease management.

This study was performed to i) compare root distribution of legume/non-legume intercrops determined by qPCR and amplicon sequencing (Meta-barcoding) to root growth of the individual species determined visually, and ii) compare the qPCR and amplicon sequencing techniques as a method for quantifying root growth of individual species grown in mixtures. Red beet (Beta vulgaris L. cv Detroit) and lucerne (Medicago sativa L. cv Creno) were grown in one-meter-high transparent tubes. Root competition was studied by direct observation on the tube surface determining root intensity (RI), root wash and extraction followed by determination of root length density (RLD), and DNA-based methods. Intercropping decreased lucerne RI strongly, whereas the RI of red beet was not affected. The determination of RLD did not allow us to distinguish species in mixed samples. However, both DNA methods showed apparently a tendency to overestimate the fraction of lucerne roots compared to the direct root observations. The discrepancy between methods was explained by the fact that visual methods provide estimates of root length while the DNA methods provide estimates of root mass. In conclusion, DNA-based estimates of species fractions in mixed root samples give valuable information on root interactions in mixed crops.

Background Quinoa (Chenopodium quinoa Willd.) is an ancient grain crop that is tolerant to abiotic stress and has favorable nutritional properties. Downy mildew is the main disease of quinoa and is caused by infections of the biotrophic oomycete Peronospora variabilis Gaüm. Since the disease causes major yield losses, identifying sources of downy mildew tolerance in genetic resources and understanding its genetic basis are important goals in quinoa breeding. Results We infected 132 South American genotypes, three Danish cultivars and the weedy relative C. album with a single isolate of P. variabilis under greenhouse conditions and observed a large variation in disease traits like severity of infection, which ranged from 5 to 83%. Linear mixed models revealed a significant effect of genotypes on disease traits with high heritabilities (0.72 to 0.81). Factors like altitude at site of origin or seed saponin content did not correlate with mildew tolerance, but stomatal width was weakly correlated with severity of infection. Despite the strong genotypic effects on mildew tolerance, genome-wide association mapping with 88 genotypes failed to identify significant marker-trait associations indicating a polygenic architecture of mildew tolerance. Conclusions The strong genetic effects on mildew tolerance allow to identify genetic resources, which are valuable sources of resistance in future quinoa breeding.

Background Soaking of sorghum seeds for six hours in an aqueous extract of Eclipta alba has been shown to increase the yield of sorghum in field experiments. The effect on yield is known to depend on field location and a mechanism involving pathogen suppression has been proposed. However, it has not been clear to which extent the same effect can be obtained by soaking of seeds in pure water (hydropriming). To address this question, fifty eight field tests were conducted comparing no treatment of seeds, hydropriming and treatment with plant extract. Experiments were distributed over three years in Burkina Faso on three locations previously showing a positive yield response to the plant extract. Results Despite strong variation across locations and years, a mean yield increase of 19.6% was found for hydropriming compared to no treatment (p Conclusion Using 6-hours of soaking, hydropriming was an inherent component of seed treatment with the E. alba extract and contributed significantly to the overall observed increase of yield and emergence. An additional yield increase was caused by factor(s) derived from the plant, E. alba, and may involve suppression of pathogenic fungi.

Hydropriming of pearl millet seeds was tested during two growing seasons in Burkina Faso. A total of 32 field experiments were distributed equally between two agro-ecological zones: The Northern zone receiving on average less than 600 mm annual precipitation and the Central zone receiving 600 to 900 mm. Hydropriming was performed by soaking of seeds in water for 6 h, followed by air-drying overnight. In the Northern zone, an increase of both emergence and yield was observed for hydroprimed seeds in both years of testing. This was reflected by a higher yield observed in 13 out of 16 field experiments, increased median yield (+159 kg/ha; p

Abies nordmanniana is a major Christmas tree species in Europe, but their uneven and prolonged growth slows down their production. By a 16S and 18S rRNA gene amplicon sequencing approach, we performed a characterization of root-associated bacterial and fungal communities for three-year-old A. nordmanniana plants collected from two nurseries in Denmark and Germany and displaying different growth patterns (small versus tall plants). Proteobacteria had the highest relative abundance at both sampling sites and plant sizes, and Ascomycota was the most abundant fungal phylum. At the order level, Acidobacteriales, Actinomycetales, Burkholderiales, Rhizobiales, and Xanthomonadales represented the bacterial core microbiome of A. nordmanniana, independently of the sampling site or plant size, while the fungal core microbiome included members of the Agaricales, Hypocreales, and Pezizales. Principal Coordinate Analysis indicated that both bacterial and fungal communities clustered according to the sampling site pointing to the significance of soil characteristics and climatic conditions for the composition of root-associated microbial communities. Major differences between communities from tall and small plants were a dominance of the potential pathogen Fusarium (Hypocreales) in the small plants from Germany, while Agaricales, that includes reported beneficial ectomycorrhizal fungi, dominated in the tall plants. An evaluation of plant root antioxidative enzyme profiles showed higher levels of the antioxidative enzymes ascorbate peroxidase, peroxidase, and superoxide dismutase in small plants compared to tall plants. We suggest that the higher antioxidative enzyme activities combined with the growth arrest phenotype indicate higher oxidative stress levels in the small plants. Additionally, the correlations between the relative abundances of specific taxa of the microbiome with the plant antioxidative enzyme profiles were established. The main result was that many more bacterial taxa correlated positively than negatively with one or more antioxidative enzyme activity. This may suggest that the ability of bacteria to increase plant antioxidative enzyme defenses is widespread.

Leaf blight and fruit rot disease caused by Phomopsis vexans is a devastating disease of brinjal. The detection of P. vexans in plant parts and seeds of brinjal can be complicated, mainly when the inoculum is present at low levels and/or overgrown by fast-growing saprophytic fungi or other seed-borne fungi. A PCR-based diagnostic method was developed with specific primers designed based on sequence data of a region consisting of the 5·8S RNA gene and internal transcribed spacers, ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of P. vexans. The efficiency and specificity of primer pairs PvexF/PvexR designed were established by PCR analysis of DNA from P. vexans strains isolated from India and fungal isolates of other genera. A single amplification product of 323-bp was detected from DNA of P. vexans isolates. No cross-reaction was observed with any of the other isolates tested. The specific primers designed and employed in PCR detected P. vexans up to 10 pg from DNA isolated from pure culture. This is the first report on the development of species-specific PCR assay for identification and detection of P. vexans. Thus, PCR-based assay developed is very specific, rapid, confirmatory and sensitive tool for the detection of pathogen P. vexans at early stages. SIGNIFICANCE AND IMPACT OF THE STUDY: Phomopsis vexans is an important seed-borne pathogenic fungus responsible for leaf blight and fruit rot in brinjal. Current detection methods, based on culture and morphological identification is time consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on sequence data of a region consisting of the 5·8S RNA gene and internal transcribed spacers, ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of P. vexans.

Eclipta alba is used as a medicinal herb in many herbal preparations including most of hair oils produced in India. The E. alba extracts are part of many Ayurvedic/herbal medicines with hepatoprotective, anti-diabetic, anti-inflammatory and anti-microbial properties. Aqueous extract, hydroalcoholic extracts and methanolic extract are reported to possess hepatoprotective, anti-diabetic, anti-inflammatory and anti-microbial properties. Studies in Burkina Faso have demonstrated that soaking seeds with 2.5% E. alba aqueous extract for 6 h improved seed quality parameters followed by increased yield in sorghum. Accordingly, it has been proposed to use E. alba for seed treatment on a larger scale. Hence it was relevant to test the toxicity of E. alba. The purpose of this study was to test the acute oral toxicity, dermal irritation and eye irritation of aqueous extract of E. alba dried leaves in Sprague Dawley rats and New Zealand white rabbits. The acute toxicity studies were carried out based on OECD guidelines 423. The highest dose administered at 2000 mg/kg body weight did not produce mortality or changes in general behaviour of the test animals indicating safety of the oral administration of aqueous E. alba extract in Sprague Dawley rats. The acute dermal irritation study in Newzealand white rabbits was investigated according to OECD test guideline No. 404. The E. alba fine powder applied to the intact left flank of female rabbit did not elicit any skin reactions at the application site of animal at any of the observation time points and hence ‘Non Irritant’ to the rabbit skin. The acute eye irritation study on Newzealand white rabbits did not cause corneal opacity, iris and conjunctivae in any of the treated animals and did not cause staining of the treated eye and is termed as ‘not irritating’ to the rabbit eyes / eye mucosa. The toxicological studies prove that the E. alba aqueous extract are safe to be used as seed treatment and can be handled safely by humans under field conditions.

Xanthomonas campestris pv. musacearum (Xcm) causing the banana Xanthomonas wilt (BXW) disease has been the main xanthomonad associated with bananas in East and Central Africa based on phenotypic and biochemical characteristics. However, biochemical methods cannot effectively distinguish between pathogenic and non-pathogenic xanthomonads. In this study, gram-negative and yellow-pigmented mucoid bacteria were isolated from BXW symptomatic and symptomless bananas collected from different parts of Uganda. Biolog, Xcm-specific (GspDm), Xanthomonas vasicola species-specific (NZ085) and Xanthomonas genus-specific (X1623) primers in PCR, and sequencing of ITS region were used to identify and characterize the isolates. Biolog tests revealed several isolates as xanthomonads. The GspDm and NZ085 primers accurately identified three isolates from diseased bananas as Xcm and these were pathogenic when re-inoculated into bananas. DNA from more isolates than those amplified by GspDm and NZ085 primers were amplified by the X1623 primers implying they are xanthomonads, these were however non-pathogenic on bananas. In the 16-23 ITS sequence based phylogeny, the pathogenic bacteria clustered together with the Xcm reference strain, while the non-pathogenic xanthomonads isolated from both BXW symptomatic and symptomless bananas clustered with group I xanthomonads. The findings reveal dynamic Xanthomonas populations in bananas, which can easily be misrepresented by only using phenotyping and biochemical tests. A combination of tools provides the most accurate identity and characterization of these plant associated bacteria. The interactions between the pathogenic and non-pathogenic xanthomonads in bananas may pave way to understanding effect of microbial interactions on BXW disease development and offer clues to biocontrol of Xcm.

Successful application of virus-induced gene silencing for functional genomics requires a virus vector that can initiate a systemic infection of the host plant. Agroinoculation of the pea early browning virus vectors pCAPE1 and pCAPE2 can establish infection in several genotypes of Medicago truncatula and can reduce target gene RNA levels to an extent that allows investigation of gene function.

Seed-priming of sorghum with an aqueous extract from the herbal plant, Eclipta alba has previously been found to increase crop yield of sorghum in Burkina Faso dependent on field location. In the present study, a 2.5% w/v antifungal extract from the desert tree, Balanites aegyptiaca, was similarly shown to increase the yield of sorghum by seed treatment. The effect was compared to the effect of E. alba extract on different types of seeds in different locations. A participatory trial including forty-six fields in three agro-ecological zones was conducted using local, farm-saved seeds. The overall effect on yield conferred by the B. aegyptiaca extract was significantly higher than the effect conferred by the E. alba extract (+31% versus +21%, p

Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae), one of the most economically important agricultural pests worldwide, is the vector of cassava mosaic geminiviruses that cause cassava mosaic disease (CMD). In East and Central Africa, a severe CMD pandemic that spread from Uganda in the late 1980s still continues to devastate cassava crops. To assess the association of distinct B. tabaci genetic groups with the CMD pandemic, mitochondrial cytochrome oxidase I gene sequences were analysed from whiteflies collected during surveys conducted from 2010 to 2013 in Tanzania. Four genetic groups – Sub-Saharan Africa 1 (SSA1), Mediterranean, Indian Ocean and East Africa 1, and a group of unknown whitefly species were identified. SSA1 comprised four subgroups: SSA1-SG1, SSA1-SG2, SSA1-SG1/2 and SSA1-SG3. SSA1-SG1 was confined to the pandemic-affected north-western parts of Tanzania whilst SSA1-SG2 and SSA1-SG3 were found in the central and eastern parts not yet affected by the pandemic. The CMD pandemic front was estimated to lie in Geita Region, north-western Tanzania, and to be spreading south-east at a rate of ca 26 km/year. The pandemic-associated B. tabaci SSA1-SG1 predominated up to 180 km ahead of the CMD front indicating that changes in whitefly population characteristics precede changes in disease characteristics.

All Bemisia tabaci individuals harbour an obligate bacterial symbiont (Portiera aleyrodidarum), and many also harbour non-essential facultative symbionts. The association of symbiotic bacteria with the various genetic groups of B. tabaci remains unknown for East Africa. This study aimed to assess any association between the various whitefly genetic groups and the endosymbionts they harbour; to investigate if a unique endosymbiont is associated with super-abundant whiteflies, and to provide baseline information on endosymbionts of whiteflies for a part of East Africa. Whiteflies collected during surveys in Tanzania were genotyped and screened for the presence of the obligate and six secondary symbionts (SS): Rickettsia (R), Hamiltonella (H), Arsenophonus (A), Wolbachia (W), Cardinium (C) and Fritschea (F). The results revealed the presence of Mediterranean (MED), East Africa 1 (EA1), Indian Ocean (IO) and Sub-Saharan Africa 1 (SSA1) genetic groups of Bemisia tabaci, with SSA1 further clustered into four sub-groups: SSA1-SG1, SSA1-SG2, SSA1-SG1/2 and SSA1-SG3. F was completely absent from all of the whiteflies tested while R was always found in double or multiple infections. In general, no particular symbiont appeared to be associated with the super-abundant SSA1-SG1 B. tabaci, although A or AC infections were common among infected individuals. The most striking feature of these super-abundant whiteflies, dominating cassava mosaic disease pandemic areas, was the high prevalence of individuals uninfected by any of the six SS tested. This study of the endosymbionts of B. tabaci in East Africa showed contrasting patterns of infection in crop and weed hosts.

An aqueous extract of the sisal agave, A. sisalana, was prepared from dried stem tissue by boiling in water. The sisal extract (SE) displayed a broad inhibition of the seed-borne mycoflora when applied to farmer-saved seeds of Sorghum bicolor. Species of Fusarium (belonging to the Gibberella fujikuroi complex) were inhibited >75 % on seeds. Accordingly, seed-borne crown rot, associated with Fusarium thapsinum was significantly (p

Unscientific use of synthetic fungicides in plant disease management has environmental ramifications, such as disease resurgence and serious health problems due to their carcinogenicity. This has prompted the identification and development of eco-friendly greener alternatives. Eclipta alba extract was evaluated for its antifungal activity in in vitro and in vivo against sorghum fungal pathogens Fusarium thapsinum, Alternaria alternata, Epicoccum sorghinum, and Curvularia lunata. The column purified methanolic extract of E. alba exhibited good antifungal activity against the target pathogens. The MIC was observed at 80 mg/mL for all tested pathogenic fungi, whereas MFC was 80 mg/mL for E. sorghinum, 100 mg/mL for F. thapsinum, A. alternata, and C. lunata. In vitro germination percentage was significantly high in seeds treated with E. alba extract (98%) over untreated control (91%). Significant disease protection of 95% was observed in greenhouse and 66% disease protection was noticed in field experiments. The efficacy of E. alba extract in field conditions was improved with the use of E. alba extract formulation. The profile of phytochemicals in E. alba methanol fractions was obtained by ultra-performance liquid chromatography (UPLC) mass spectroscopy. The [M-H]&minus, at m/z 313.3, m/z 797.9, and m/z 269.0 revealed the presence of wedelolactone, eclalbasaponin II, and apigenin, respectively. The H-nuclear magnetic resonance spectroscopy (1H-NMR) chemical shift value supported the findings of the mass spectrometry. The results highlighted the possible use of E. alba methanolic extract as alternative to chemical fungicide in sorghum disease management.

A PCR-based system was developed to reliably and robustly identify group I and II members of the genus Xanthomonas. Primer sets developed from three gene targets namely fyuA, ITS and gumD were evaluated in the study. Primer sets were evaluated using DNA extracted from 45 Xanthomonas strains representing 25 species broadly covering the genus. Fifteen non-Xanthomonas strains of plant-associated bacteria including phylogenetically closely related species Stenotrophomonas maltophilia and Xylella fastidiosa were also tested. The primers targeting fyuA amplified DNA from all xanthomonads except X. theicola, while the ITS primers amplified a DNA fragment of 254 bp in all 45 Xanthomonas strains; whereas no amplification was observed for non-xanthomonads. The gumD primers allowed efficient amplification of DNA in 38 out of 39 isolates from Group II, whereas no or very weak amplification occurred with DNA from Group I members. Internal controls of primers targeting bacterial 16S rDNA or plant 26S mitochondrial rDNA were successfully applied in multiplex PCRs for testing bacterial cultures or plant tissue, respectively. The findings give us a PCR based approach that can reliably and effectively differentiate xanthomonads from non-xanthomonads as well as separating the strains belonging to the two described groups of the genus Xanthomonas. The study thus offers valuable tools for disease surveillance and management. It can effectively be applied in rapid assessment of new disease occurrences, for which no specific detection tools could be in place.

Aims: To develop a sampling procedure for PCR-based screening of bacterial leaf spot (BLS)-causing xanthomonads without DNA extraction from infected tomato plants. Place and Duration of Study: University of Copenhagen, Denmark and Sokoine University of Agriculture, Morogoro, Tanzania between July 2008 and November 2010. Methodology: Flinders Technology Associates (FTA) plant cards and Chromatography paper or Whatman paper strips (WPS) were spotted with bacterial suspensions from 24h-old cultures from reference strains of BLS-causing xanthomonads, or sap obtained by grinding or hand maceration of plant tissue, were used as templates in PCR reactions or isolation of live bacterial cells on Nutrient agar (NA) media. Samples were tested by PCR with Xan 7 genus/-specific Xanthomonas primers or in multiplex with 26S rRNA primers. Isolation of bacteria was done by streaking aliquots of 75 μl of a suspension from a disc Research Article British Biotechnology Journal, 3(4): 556-574, 2013 557 (2-mm-punch by Harris Micro Punch®) in triplicate, removed from each of the FTA plant card and WPS onto NA media. Results: The FTA plant card spotted with pure cultures of reference strains of xanthomonads and sap from grinding or direct maceration of plant tissue resulted in more clear PCR bands (402 bp) and (594 bp of rRNA gene in multiplex) than the WPS samples. Sensitivity of detection by the FTA paper-based PCR was ≈ 5.0 x 10, while that of the WPS was > 1.0 x 10 CFU/ml. The WPS (but not the FTA) was proved to be useful for saving living bacteria cells for up to one week of storage at ambient temperatures. Conclusion: Both FTA plant card and WPS can be used for PCR detection of BLScausing xanthomonads in tomato. However, the FTA plant card is recommended as it produced clearer PCR products than WPS. WPS is recommended for experiments requiring isolation of live bacterial cells on NA media.

Tomato ( Solanum lycopersicum L.) seeds habour unique bacterial community that can be pathogenic or beneficial to their host. Xanthomonas causing bacterial leaf spot (BLSX) on tomato and other yellow-pigmented xanthomonads-like bacteria (XLB) that closely resemble BLSX were obtained from tomato seeds collected from Northern, Central and Southern highland regions of Tanzania. A total of 73 strains were isolated from 52 seed samples of 15 tomato cultivars. Results obtained with Biolog and sequence analysis of the 16S rRNA gene showed that samples originating from Central Tanzania harbored the most diverse populations of XLB and BLSX as compared to Northern and Southern Tanzania. The predominant bacterial genera in tomato seeds were Stenotrophomonas , Sphingomonas , Chryseobacterium , Xanthomonas , Pantoea and Flavobacterium . All strains identified by Biolog as Xanthomonas with exception of Xanthomonas campestris pv. malvacearum , were pathogenic on tomato and pepper plants. Strains identified by Biolog as Sphingomonas sanguinis and Sphingomonas terrae also incited black rot symptoms on pepper leaves. However, bacterial strains belonging to the genus Stenotrophomonas , Chryseobacterium , Pantoea and Flavobacterium were not pathogenic on tomato and pepper. Phylogenetic analysis showed that strains of the genus Xanthomonas are more closely related to Stenotrophomonas and Pantoea compared to the other bacterial genera found in tomato seeds. Key words: Xanthomonas , yellow-pigmented bacteria, seed, tomato, phylogeny.

Aims: To develop a highly sensitive and rapid protocol for simultaneous detection and differentiation of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in pepper and tomato. In this study, we use the multiplex PCR technique to detect dual infection of these two viruses. Methods and Results: A multiplex RT–PCR method consisting of one-tube reaction with two primer pairs targeted to replicase genes was developed to simultaneously detect TMV and ToMV in seed samples of pepper and tomato. Specific primers were designed from conserved regions of each of the virus genomes, and their specificity was confirmed by sequencing PCR products. RT–PCR detected up to 10−6 dilution of total RNA extracted from infected leaves. Multiplex RT–PCR revealed the presence of both TMV and ToMV in three of 18 seed samples of tomato and one of 18 seed samples of pepper. Conclusions: The multiplex PCR assay was a cost effective, quick diagnostic technique, which was helpful in differentiating TMV and ToMV accurately. Significance and Impact of the Study: The multiplex PCR assay described in this study is a valuable tool for plant pathology and basic research studies. This method may facilitate better recognition and distinction of TMV and ToMV in both pepper and tomato.

Transgenic crops are now grown commercially in 25 countries worldwide. Although pathogens represent major constraints for the growth of many crops, only a tiny proportion of these transgenic crops carry disease resistance traits. Nevertheless, transgenic disease-resistant plants represent approximately 10% of the total number of approved field trials in North America, a proportion that has remained constant for 15 years. In this review, we explore the socioeconomic and biological reasons for the paradox that although technically useful solutions now exist for providing transgenic disease resistance, very few new crops have been introduced to the global market. For bacteria and fungi, the majority of transgenic crops in trials express antimicrobial proteins. For viruses, three-quarters of the transgenics express coat protein (CP) genes. There is a notable trend toward more biologically sophisticated solutions involving components of signal transduction pathways regulating plant defenses. For viruses, RNA interference is increasingly being used.

Eukaryotic translation initiation factors (eIFs) play a central role in potyviral infection. Accordingly, mutations in the gene encoding eIF4E have been identified as a source of recessive resistance in several plant species. In common bean, Phaseolus vulgaris, four recessive genes, bc-1, bc-2, bc-3 and bc-u, have been proposed to control resistance to the potyviruses Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus. In order to identify molecular entities for these genes, we cloned and sequenced P. vulgaris homologues of genes encoding the eIF proteins eIF4E, eIF(iso)4E and nCBP. Bean genotypes reported to carry bc-3 resistance were found specifically to carry non-silent mutations at codons 53, 65, 76 and 111 in eIF4E. This set of mutations closely resembled a pattern of eIF4E mutations determining potyvirus resistance in other plant species. The segregation of BCMV resistance and eIF4E genotype was subsequently analysed in an F2 population derived from the P. vulgaris all-susceptible genotype and a genotype carrying bc-3. F2 plants homozygous for the eIF4E mutant allele were found to display at least the same level of resistance to BCMV as the parental resistant genotype. At 6 weeks after inoculation, all F2 plants found to be BCMV negative by enzyme-linked immunosorbent assay were found to be homozygous for the mutant eIF4E allele. In F3 plants homozygous for the mutated allele, virus resistance was subsequently found to be stably maintained. In conclusion, allelic eIF4E appears to be associated with a major component of potyvirus resistance present in bc-3 genotypes of bean.

Virus-induced gene silencing (VIGS) has become an important reverse genetics tool for functional genomics. VIGS vectors based on Pea early browning virus (PEBV, genus Tobravirus) and Bean pod mottle virus (genus Comovirus) are available for the legume species Pisum sativum and Glycine max, respectively. With the aim of extending the application of the PEBV VIGS vector to other legumes, we examined susceptibility of 99 accessions representing 24 legume species including 21 accessions of Medicago truncatula and 38 accessions Lotus japonicus. Infectivity of PEBV was tested by agro-inoculation with a vector carrying the complete β-glucuronidase (GUS) coding sequence. In situ histochemical staining analysis indicated that 4 of 21 M. truncatula and three of three Lathyrus odorata accessions were infected systemically by GUS tagged PEBV, while none of 38 L. japonicus accessions displayed GUS staining of either inoculated or uninoculated leaves. Agro-inoculation of plants representing PEBV-GUS susceptible M. truncatula and L. odorata accessions with PEBV carrying a fragment of Phytoene desaturase (PDS) resulted in development of a bleaching phenotype suggesting a down-regulation of PDS expression. In M. truncatula this was supported by quantification of PDS mRNA levels by real-time PCR Udgivelsesdato: August Virus-induced gene silencing (VIGS) has become an important reverse genetics tool for functional genomics. VIGS vectors based on Pea early browning virus (PEBV, genus Tobravirus) and Bean pod mottle virus (genus Comovirus) are available for the legume species Pisum sativum and Glycine max, respectively. With the aim of extending the application of the PEBV VIGS vector to other legumes, we examined susceptibility of 99 accessions representing 24 legume species including 21 accessions of Medicago truncatula and 38 accessions Lotus japonicus. Infectivity of PEBV was tested by agro-inoculation with a vector carrying the complete β-glucuronidase (GUS) coding sequence. In situ histochemical staining analysis indicated that 4 of 21 M. truncatula and three of three Lathyrus odorata accessions were infected systemically by GUS tagged PEBV, while none of 38 L. japonicus accessions displayed GUS staining of either inoculated or uninoculated leaves. Agro-inoculation of plants representing PEBV-GUS susceptible M. truncatula and L. odorata accessions with PEBV carrying a fragment of Phytoene desaturase (PDS) resulted in development of a bleaching phenotype suggesting a down-regulation of PDS expression. In M. truncatula this was supported by quantification of PDS mRNA levels by real-time PCR

Insect and herbicide-resistant plants are the most widely grown transgenics in agricultural production. No strategy using genetically engineered plants for disease resistance has had a comparable impact. Why is this? What are the prospects for introducing transgenic disease resistant plants to agriculture? We review the biological background for strategies used to make disease resistant GM crops, illustrate examples of these different strategies and discuss future prospects.

Herbaria collections containing plants with disease symptoms are highly valuable, and they are often the only way to investigate outbreaks and epidemics from the past as the number of viable isolates in culture collections is often limited. Species belonging to the Colletotrichum acutatum complex infect a range of important crops. As members of the C. acutatum complex are easily confused with other Colletotrichum species, molecular methods are central for the correct identification. We performed molecular analyses on 21 herbaria specimens, displaying anthracnose symptoms, collected in Norway and Denmark before the first confirmed findings of C. acutatum complex members in this region. Sequencing parts of the fungal ITS regions showed that members of the species complex were present in 13 of the 21 specimens collected in different parts of Norway and Denmark between 1948 and 1991, representing seven plant hosts (three cherry species, apple, raspberry and rhododendron). This is the first time herbarium specimens have been used to study these pathogens under Nordic conditions. Differences in the ITS sequences suggest the presence of different genotypes within the complex, indicating a well-established population.

Summary Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studies of gene function. However, efficient VIGS has only been accomplished in a few plant species. In order to extend the application of VIGS, we examined whether a VIGS vector based on Pea early browning virus (PEBV) would produce recognizable phenotypes in Pisum sativum. A plasmid vector of PEBV was modified to allow agro-inoculation and insertion of heterologous sequences. cDNA fragments of the P. sativum phytoene desaturase (PDS), LEAFY (LFY) and KORRIGAN1 (KOR1) homologues were inserted into the PEBV RNA2 vector, replacing the genes required for nematode transmission. Pisum sativum inoculated with PEBV carrying a fragment of PsPDS developed characteristic photo-bleached leaves and this phenotype was associated with a significant reduction in PsPDS mRNA. The P. sativum homologue of LFY is known as UNIFOLIATA (UNI). Plants inoculated with PEBV carrying a fragment of UNI developed distorted flowers and leaves with modified architecture, which are also observed in UNI-mutants. In Arabidopsis thaliana, the KOR1-mutant is characterized by an extreme dwarf phenotype. Pisum sativum plants inoculated with PEBV carrying a fragment of PsKOR1 displayed a significant reduction in height and inhibition of root growth. The PEBV VIGS vector did not affect the ability of P. sativum to flower, set seeds, and form nodules characteristic of symbiosis with rhizobium. These results suggest that the PEBV vector can be applied to functional genomics in a legume species to study genes involved in a wide range of biological processes.

[This corrects the article DOI: 10.1371/journal.pone.0176469.].

We have previously shown that an N-glycosylation site of N306 of HIV-1 gp120 is not necessary for the HIV-1 infectivity but protects HIV-1 from neutralising antibodies. In contrast Nakayama et al. [FEBS Lett. (1998) 426, 367–372], using a virus with an identical V3 region, suggested that elimination of this particular glycan reduced the ability of T-tropic HIV to bind to CXCR4 and hence its ability to infect T cell lines. We therefore re-examined the ability of a mutant virus, lacking the N306 glycan, to replicate in various types of cells and found no change in co-receptor usage for mutant virus. The ability of mutant virus to replicate or to induce syncytia in infected cells was similar to that of wild type virus. These results corroborate our original observation, confirming that the induced mutation in the N306 glycosylation site neither impairs nor improves the ability of mutant virus to replicate in permissive cells.

An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected to elicit antibodies preferentially neutralizing mutant variants of HIV-BRU lacking the N306 glycan. Therefore, two guinea pigs were immunized with monomeric wild-type HIV-BRU gp120 possessing the N306 glycan and immune sera were tested for neutralization against target viruses HIV-BRU, -A308, and -A308T321. HIV-A308 and HIV-A308T321 lack the N306 glycan; HIV-A308T321 contains an additional mutation at the tip of V3 rendering it resistant to MAb binding at this epitope. Both immune sera preferentially neutralized the two mutant virus variants lacking the N306 glycan, with a 10- to 20-fold increase in neutralization titer compared with the wild-type HIV-BRU. Thus, immunization with monomeric HIV-BRU gp120 elicited antibodies preferentially neutralizing HIV variants lacking the N306 glycan. In addition to antibodies directed against the tip of V3, other antibodies directed against epitopes shielded by the N306 glycan on the envelope oligomer were elicited by the immunization, as demonstrated by the ability of the immune sera to neutralize HIV-A308T321. One such epitope was overlapping the NEA-9284 epitope located at the amino-terminal flank of the V3 loop. Our results demonstrate that monomeric gp120 contains immunogenic structures inaccessible on the envelope oligomer. The limited ability of recombinant gp120 vaccines to induce neutralizing antibodies against primary isolates may thus not exclusively reflect genetic variation.

This paper presents a mathematical analysis of the criteria for gene therapy of T helper cells to have a clinical effect on HIV infection. The analysis indicates that for such a therapy to be successful, it must protect the transduced cells against HIV-induced death. The transduced cells will not survive as a population if the gene therapy only blocks the spread of virus from transduced cells that become infected. The analysis also suggests that the degree of protection against disease-related cell death provided by the gene therapy is more important than the fraction cells that is initially transduced. If only a small fraction of the cells can be transduced, transduction of T helper cells and transduction of haematopoietic progenitor cells will result in the same steady-state level of transduced T helper cells. For gene therapy to be efficient against HIV infection, our analysis suggests that a 100% protection against viral escape must be obtained. The study also suggests that a gene therapy against HIV infection should be designed to give the transduced cells a partial but not necessarily total protection against HIV-induced cell death, and to avoid the production of viral mutants insensitive to the gene therapy.

We investigated if complement-mediated enhancement of HIV infection occurs in peripheral blood mononuclear cells (PBMC). In 7 experiments, we evaluated the effect of human complement on HIVIIIB infection in vitro. We measured HIV antigen production on day 4 and found that pre-incubation of HIV with complement led to enhanced production of antigen with a median enhancement of 2.5-fold (range 1.1-6.8). This complement-mediated increase in antigen production was statistically significant (p0.02). Complement-mediated enhancement of HIV infection was also tested in CD4 cells enriched from PBMC, and CD4 cells persistently gave higher levels of infection enhancement than PBMC. Thus, CD4 cells appear to be sufficient for complement-mediated enhancement of HIV infection to occur. In addition, we tested if it was possible to detect complement-mediated enhancement of primary HIV isolates in PBMC. We tested 3 isolates and found only a minor effect on antigen production (median enhancement 1.2-fold, range 0.6-1.5). Furthermore, addition of HIV-specific antibodies in combination with complement resulted in enhanced antigen production in 2/3 sera tested. However, the combination of complement and antibodies resulted in only a minor increase in enhancement of HIV infection compared to that obtained with complement alone. Finally, we found evidence of complement-mediated enhancement of HIV infection in resting PBMC. In conclusion, we demonstrated that complement-mediated enhancement of HIV infection does occur in vitro in PBMC.

A 15-mer C-5 propyne modified phosphorothioate oligodeoxynucleotide antisense to rev was approximately 5-fold more effective in providing viral inhibition compared to a 28-mer unmodified phosphorothioate oligodeoxynucleotide targeted to the same sequence and previously shown to inhibit HIV-1 in a sequence-dependent manner. The antiviral effect was obtained by lipofection or simple addition of 0.2–1 μM modified oligodeoxynucleotide to the culture medium of H9 cells chronically infected with the HIV-1 LAI isolate of human immunodeficiency virus type 1. We conclude that C-5 propyne oligodeoxynucleotides in accordance with previous findings by others are superior to unmodified phosphorothioates in providing inhibition of HIV-1 in a sequence-dependent manner and that this inhibition can be conferred by oligodeoxynucleotides in free solution.

Alternaria helianthi is an important seed-borne pathogenic fungus responsible for blight disease in sunflower. The current detection methods, which are based on culture and morphological identification, are time-consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on the sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers—ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of A. helianthi. The specificity of the primer pairs AhN1F and AhN1R designed was verified by PCR analysis of DNA from 18 Alternaria helianthi strains isolated from India, 14 non-target Alternaria spp. and 11 fungal isolates of other genera. A single amplification product of 357-bp was detected from DNA of A. helianthi isolates. No cross-reaction was observed with any of the other isolates tested. The detection limit of the PCR method was of 10 pg from template DNA. The primers could also detect the pathogen in infected sunflower seed. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of A. helianthi. This is the first report of an A. helianthi-specific primer set.

Virus-induced gene silencing (VIGS) can provide a shortcut to plants with altered expression of specific genes. Here, we report that VIGS of the Nodule inception gene (Nin) can alter the nodulation phenotype and Nin gene expression in Pisum sativum. PsNin was chosen as target because of the distinct non-nodulating phenotype of nin mutants in P. sativum, Lotus japonicus, and Medicago truncatula. The vector based on Pea early browning virus (PEBV) was engineered to carry one of three nonoverlapping fragments (PsNinA, PsNinB, and PsNinC) derived from the PsNin cDNA. Vector inoculation was mediated by agroinfiltration and, 2 weeks later, a Rhizobium leguminosarum bv. viceae culture was added in order to induce root nodulation. At this time point, it was estimated that systemic silencing was established because leaves of reference plants inoculated with PEBV carrying a fragment of Phytoene desaturase displayed photo bleaching. Three weeks after Rhizobium spp. application, plants inoculated with a control vector nodulated normally, whereas nodulation was almost eliminated in plants inoculated with a vector carrying PsNinA and PsNinC. For plants inoculated with a vector carrying PsNinB, nodulation was reduced by at least 45%. Down-regulation of PsNin transcripts in plants inoculated with vectors carrying PsNin cDNA fragments was confirmed and these plants displayed a relative increase in the root/shoot ratio, as expected if nitrogen fixation had been impaired Virus-induced gene silencing (VIGS) can provide a shortcut to plants with altered expression of specific genes. Here, we report that VIGS of the Nodule inception gene (Nin) can alter the nodulation phenotype and Nin gene expression in Pisum sativum. PsNin was chosen as target because of the distinct non-nodulating phenotype of nin mutants in P. sativum, Lotus japonicus, and Medicago truncatula. The vector based on Pea early browning virus (PEBV) was engineered to carry one of three nonoverlapping fragments (PsNinA, PsNinB, and PsNinC) derived from the PsNin cDNA. Vector inoculation was mediated by agroinfiltration and, 2 weeks later, a Rhizobium leguminosarum bv. viceae culture was added in order to induce root nodulation. At this time point, it was estimated that systemic silencing was established because leaves of reference plants inoculated with PEBV carrying a fragment of Phytoene desaturase displayed photo bleaching. Three weeks after Rhizobium spp. application, plants inoculated with a control vector nodulated normally, whereas nodulation was almost eliminated in plants inoculated with a vector carrying PsNinA and PsNinC. For plants inoculated with a vector carrying PsNinB, nodulation was reduced by at least 45%. Down-regulation of PsNin transcripts in plants inoculated with vectors carrying PsNin cDNA fragments was confirmed and these plants displayed a relative increase in the root/shoot ratio, as expected if nitrogen fixation had been impaired

Some DNA fragments are difficult to clone in Escherichia coli by standard methods. It has been speculated that unintended transcription and translation result in expression of proteins that are toxic to the bacteria. This problem is frequently observed during assembly of infectious full-length virus clones. If the clone is constructed for transcription in vivo, interrupting the virus sequence with an intron can solve the toxicity problem. The AU-rich introns generally contain many stop codons, which interrupt translation in E. coli, while the intron sequence is precisely eliminated from the virus sequence in the plant nucleus. The resulting RNA, which enters the cytoplasm, is identical to the virus sequence and can initiate infection.

Some DNA fragments are difficult to clone in Escherichia coli by standard methods. It has been speculated that unintended transcription and translation result in expression of proteins that are toxic to the bacteria. This problem is frequently observed during assembly of infectious full-length virus clones. If the clone is constructed for transcription in vivo, interrupting the virus sequence with an intron can solve the toxicity problem. The AU-rich introns generally contain many stop codons, which interrupt translation in E. coli, while the intron sequence is precisely eliminated from the virus sequence in the plant nucleus. The resulting RNA, which enters the cytoplasm, is identical to the virus sequence and can initiate infection Some DNA fragments are difficult to clone in Escherichia coli by standard methods. It has been speculated that unintended transcription and translation result in expression of proteins that are toxic to the bacteria. This problem is frequently observed during assembly of infectious full-length virus clones. If the clone is constructed for transcription in vivo, interrupting the virus sequence with an intron can solve the toxicity problem. The AU-rich introns generally contain many stop codons, which interrupt translation in E. coli, while the intron sequence is precisely eliminated from the virus sequence in the plant nucleus. The resulting RNA, which enters the cytoplasm, is identical to the virus sequence and can initiate infection

Udgivelsesdato: September Pathogenicity of two sequenced isolates of Bean yellow mosaic virus (BYMV) was established on genotypes of Pisum sativum L. reported to carry resistance genes to BYMV and other potyviruses. Resistance to the white lupin strain of BYMV (BYMV-W) is inherited as a recessive gene named wlv that maps to linkage group VI together with other Potyvirus resistances. One of these, sbm1, confers resistance to strains of Pea seedborne mosaic virus and previously has been identified as a mutant allele of the eukaryotic translation initiation factor 4E gene (eIF4E). Sequence comparison of eIF4E from BYMV-W-susceptible and â€"resistant P. sativum genotypes revealed a polymorphism correlating with the resistance profile. Expression of eIF4E from susceptible plants in resistant plants facilitated BYMV-W infection in inoculated leaves. When cDNA of BYMV-W was agroinoculated, resistance mediated by the wlv gene frequently was overcome, and virus from these plants had a codon change causing an Arg to His change at position 116 of the predicted viral genome-linked protein (VPg). Accordingly, plants carrying the wlv resistance gene were infected upon inoculation with BYMV-W derived from cDNA with a His codon at position 116 of the VPg coding region. These results suggested that VPg determined pathogenicity on plants carrying the wlv resistance gene and that wlv corresponded to the sbm1 allele of eIF4E

A fourth pathotype of Pea seedborne mosaic virus, a member of the genus Potyvirus, was identified by analysis of the infection profile on a panel of Pisum sativum lines. The new pathotype, designated P-3, was able to overcome resistance specified by the sbm-1 resistance gene but could not overcome resistance specified by the sbm-2 resistance gene. This infection profile distinguished P-3 from previously described pathotypes, P-1, P-2, and P-4. Analysis of chimeric viruses demonstrated that properties of the P3-6k1- and viral genome-linked protein (VPg)-coding regions accounted for the infection profile of the new pathotype.

Pea seed-borne mosaic potyvirus (PSbMV) isolates are divided into pathotypes P-1, P-2, and P-4 according to their infection profile on a panel of Pisum sativum lines. P. sativum PI 269818 is resistant to P-1 and P-2 isolates and is susceptible to P-4 isolates. Resistance to P-1 is inherited as a single recessive gene, denoted sbm-1, and the pathogenicity determinant has previously been mapped to the virus-coded protein VPg. In the cultivar Bonneville, a second recessive gene, sbm-2, confers specific resistance to P-2. By exchanging cistrons between a P-2 and a P-4 isolate, the P3-6k1 cistron was identified as the PSbMV host-specific pathogenicity determinant on Bonneville. Exchange of P3-6k1 did not affect infection on PI 269818, and infection of Bonneville was not altered by substitution of the VPg cistron, indicating that P3-6k1 and VPg are independent determinants of pathotype-specific infectivity. On PI 269818 the pathogenicity determinant of both P-1 and P-2 mapped to the N terminus of VPg. This suggests that VPg from the P-1 and P-2 isolates are functionally similar on this host and that resistance to P-1 and P-2 in PI 269818 may operate by the same mechanism. Identification of VPg– sbm-1 and P3-6k1– sbm-2 as independent pairs of genetic interactors between PSbMV and P. sativum provides a simple explanation of the three known pathotypes of PSbMV. Furthermore, analysis of β-glucuronidase-tagged P-2 virus indicated that sbm-2 resistance affected an early step in infection, implying that the P3-6k1 region plays a critical role in potyvirus replication or cell-to-cell movement.

In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an env transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb neutralization gradually increased. Virus neutralization by virion aggregation was minimal, as MAb binding to HIV-1 Env did not interfere with an AMLV Env-mediated infection by HIV-1(AMLV/HIV-1) pseudotypes of CD4 − HEK293 cells. MAb neutralization of chimeric virions could be described as a third-order function of the proportion of Env antigen refractory to MAb binding. This scenario is consistent with the Env oligomer constituting the minimal functional unit and neutralization occurring incrementally as each Env oligomer binds MAb. Alternatively, the data could be fit to a sigmoid function. Thus, these data could not exclude the existence of a threshold for neutralization. However, results from MAb neutralization of chimeric virus containing wild-type Env and Env defective in CD4 binding was readily explained by a model of incremental MAb neutralization. In summary, the data indicate that MAb neutralization of T-cell line-adapted HIV-1 is incremental rather than all or none and that each MAb binding an Env oligomer reduces the likelihood of infection.

An amino acid substitution (D --> K) in the C3 region of HIV-1 gp120 has previously been shown to inhibit binding of virions to CD4+ cells. We have introduced the same mutation into the HIV-1 isolate LAV-I(BRU), in which the mutation is denoted D373K. Here we show that the D373K envelope protein is processed and incorporated into virus particles, but that D373K virions have no detectable infectivity (below 0.1% relative to wild type). When D373K and the wild-type envelope gene were cotransfected in 293 cells at a 4:1 ratio, the resultant infectivity of the HIV-1 supernatant was reduced more than 100-fold. When the same ratio of plasmids was tested in COS-1 cells the inhibition of HIV-1 was an order of magnitude less than observed in 293 cells. COS-1 and 293 cells differed in that only 293 cells displayed saturation of virus production with respect to the envelope protein. Our data fit a simple model: when virion formation is saturated with envelope protein, expression and incorporation of a defective envelope protein imply a corresponding dilution of wild-type protein on the surface of virions. The cooperative function of wild-type envelope proteins is subsequently compromised, and a trans-dominant inhibition of virus infectivity is observed.