The evaluation of rhizomania resistant sugar beet for the UK

Sugar beet rhizomania disease, caused by Beet necrotic yellow vein virus and transmitted by the soil‐borne parasite Polymyxa betae, was first recorded in the UK in 1987. Recently, breeding lines and cultivars with partial resistance to the virus derived from the ‘Holly’ source of resistance have been developed and their suitability for use under UK conditions is explored in this paper. Virus multiplication in the roots of resistant lines exposed to severe disease pressure in glasshouse tests, when quantified by ELISA, was less than one third of that in susceptible controls. More recently developed resistant lines had a lower virus content, on average, largely due to a reduced frequency of susceptible individuals. There was no evidence for resistance to the vector, P. betae, in virus resistant lines. However, the proportion of viruliferous P. betae resting spores in the roots, estimated using the most probable number (MPN) technique, was reduced by at least one third in resistant lines compared with the most susceptible control. A novel line, containing an additional gene to that in ‘Holly’, was the most effective, reducing the infection level to 3% of that in the susceptible control. In two field experiments on severely infested sites, the rate of infection of a resistant line, when assessed by ELISA, was reduced by half compared with a susceptible cultivar and sugar yields of resistant lines were consistently 2–3 times higher than those of susceptible cultivars. In 41 trials on rhizomania‐free sites, several recently introduced resistant lines exhibited sugar yields and agronomic performance comparable to that of three selected high yielding, susceptible cultivars. Results are discussed in relation to the specific UK requirements for rhizomania resistant cultivars. One resistant line, Beta 805 (cv. Concept), fulfilled the requirements for widespread use to control the disease.     

Der mit der Versuchsdauer anwachsende Resistenzfaktor

Abstract

Pseudomonas spp. strains capable of inducing systemic resistance were applied to sugarcane by sett treatment followed by soil applications in the field. Later the fungal pathogen Colletotrichum falcatum causing red rot disease of sugarcane was inoculated in the treated canes and its colonization was assessed by ELISA at different nodal positions above the point of inoculation. Studies with three cvs showed a significant variation in pathogen colonization only in disease susceptible cv CoC 671 and not with cvs Co 8021 and BO 91, moderately susceptible and moderately resistant to the disease, respectively. In further studies when pathogen colonization was assessed on the entire stalks of cv CoC 671, the pathogen titre was significantly reduced from three nodes upwards in the treated canes. In the upper nodes no pathogen colonization was noticed in bacteria-treated canes, whereas in the control all the nodes recorded higher titre for pathogen infection. Incorporation of chitin in the talc formulation caused further reduction in fungal colonization in the stalks.     

Effect of Ploidy and Flowering Type of Red Clover Cultivars and of Isolate Origin on Severity of Clover Rot, Sclerotinia trifoliorum

Two complementary experiments were conducted in a controlled environment to elucidate the interactions between the fungus Sclerotinia trifoliorum Erikss. and red clover (Trifolium pratense L.). In one of these studies, two hardened diploid red clover cultivars (cvs) were inoculated with 20 isolates of S. trifoliorum of various geographic origins. In the other study, 20 red clover cvs, diploid or tetraploid, including late and medium‐late flowering types, were inoculated with two isolates of the fungus. Prior to inoculation, some plants were hardened by subjecting them to a low temperature and light treatment mimicking autumn conditions. Late flowering cvs were found more resistant than medium‐late ones. Isolates collected in the northern region, where late cvs are grown, were significantly more aggressive than isolates from southern locations, where medium‐late cvs are more prevalent. Such an adaptation has not previously been reported for this fungus. This is the first report concerning flowering type and resistance in red clover. Tetraploids were generally not more resistant than diploids. A hardening procedure for red clover plants was found to be a prerequisite for detecting the differences in disease resistance.     

Genetic Analysis of Resistance Gene Analogues from a Sugarcane Cultivar Resistant to Red Rot Disease

One of the important approaches for disease control in sugarcane is to develop a disease‐resistant variety; this may be accomplished through identification of resistance genes in sugarcane. In this study, PCR primers targeting the conserved motifs of the nucleotide‐binding site (NBS) class and kinase class of the resistance gene analogues (RGAs) were used to amplify the RGAs from a red rot‐resistant sugarcane cultivar (Saccharum spp. hybrid) HSF 240. Upon subcloning and sequencing, fifteen putative RGAs were identified. These RGAs shared 63–98% identity to the reported disease‐resistant genes in the NCBI GenBank database. Deduced amino acid sequences also showed the presence of expected conserved domains characteristic of RGAs. Phylogenetic analysis indicated that these RGAs clustered with R genes from other plant species. The findings will be useful for studying disease‐resistant genes in sugarcane.     

Genes tagging and molecular diversity of red rot susceptible/tolerant sugarcane hybrids using c-DNA and unigene derived markers

Sugarcane is an important international commodity as a valuable agricultural crop especially in tropical and subtropical countries. Two bulked DNA used to screen polymorphic primers from commercial hybrids (varieties) with moderately resistant and highly susceptible to red rot disease. Among 145 simple sequence repeat and unigene primers screened, 37 (25%) were found to be highly robust and polymorphic with Polymorphism Information Content values ranging from 0.50 to 1.00 with the mean value of 0.82. Among these microsatellites, twenty one were used in the study of genetic relationships and marker identification in sugarcane varieties for red rot resistance. A total of 105 polymorphic DNA bands were identified, with their fragment size ranging from 54 to 1,280 bp. Jaccard’s similarity coefficient value recorded between closely related hybrids was 0.986 while lowest coefficient value of 0.341 was detected with distantly related hybrids. The average similarity coefficient among these hybrids was 0.663. Cluster analysis resulted in a dendrogram with two major clusters separating the moderately resistant varieties from highly susceptible varieties. Three group specific fragments amplified by unigene Saccharum microsatellite primers viz; two markers UGSM316₈₅₀ and UGSM316₆₀ were closely associated with moderately resistant varieties by appearing bands in this region but the bands were absent in highly susceptible varieties. Similarly UGSM316₄₀₀ marker was tightly linked with highly susceptible varieties by amplifying uniformly in sugarcane varieties showing highly susceptible reaction to red rot but it was absent in moderately resistant varietal groups. Validation of red rot resistance/susceptibility associated markers on a group of different mapping populations for red rot resistant/susceptible traits is in progress.     

Development of a bioassay to assess resistance to Fusarium oxysporum (Schlecht.) in asparagus (Asparagus officinalis L.)

Fusarium oxysporum is one of the major pathogens causing root and crown rot in asparagus. Breeding of cultivars resistant to F. oxysporum would be the most efficient strategy for pathogen control. In this study, a bioassay was developed for screening seedling resistance. The non‐destructive bioassay comprises inoculation with a highly aggressive F. oxysporum isolate, incubation in a climate chamber and quantification of disease symptoms by a digital image analysing system and a PTA‐ELISA. This bioassay is simple to implement and demonstrated high reproducibility. Subsequently, it was used to determine the resistance behaviour of 16 asparagus genotypes to F. oxysporum. The asparagus cultivars revealed different levels of susceptibility, whereas the wild relative A. densiflorus was confirmed to be resistant.     

Poxviral Disease in Red Squirrels Sciurus vulgaris in the UK: Spatial and Temporal Trends of an Emerging Threat

The squirrel poxvirus (SQPV) is the probable mediator of apparent competition between the introduced invading gray squirrel (Sciurus carolinensis) and the red squirrel (Sciurus vulgaris) in the UK, and modeling studies have shown that this viral disease has had a significant impact on the decline of the red squirrel in the UK. However, given our limited understanding of the epidemiology of the disease, and more generally the effects of invasive species on parasite ecology, there is a need to investigate the transmission dynamics and the relative pathogenicity of the virus between species. We aimed to increase our knowledge of these processes through an empirical study in which we: (i) used pathological signs and transmission electron microscopy (TEM) to diagnose SQPV disease in red squirrels found dead during scanning surveillance between 1993 and 2005; (ii) detected antibody to SQPV using an enzyme-linked immunosorbent assay (ELISA) in the same animals; and (iii) mapped cases of the disease, and the gray squirrel distribution, using a geographical information system. We analyzed the distribution of cases of SQPV disease according to woodland type, a measure of squirrel density. SQPV disease occurred only in areas of England also inhabited by seropositive gray squirrels, and as the geographical range of gray squirrels expanded, SQPV disease occurred in these new gray squirrel habitats, supporting a role for the gray squirrel as a reservoir host of the virus. There was a delay between the establishment of invading gray squirrels and cases of the disease in red squirrels which implies gray squirrels must reach a threshold number or density before the virus is transmitted to red squirrels. The spatial and temporal trend in SQPV disease outbreaks suggested that SQPV disease will have a significant effect on Scottish populations of red squirrels within 25 years. The even spread of cases of disease across months suggested a direct rather than vector-borne transmission route is more likely. Eight juvenile and sub-adult free-living red squirrels apparently survived exposure to SQPV by mounting an immune response, the first evidence of immunity to SQPV in free-living red squirrels, which possibly suggests a changing host-parasite relationship and that the use of a vaccine may be an effective management tool to protect remnant red squirrel populations.     

Detection of Colletotrichum falcatum causing red rot of sugarcane by enzyme-linked immunosorbent assay

Red rot disease of sugarcane caused by Colletotrichum falcatum Went is one of the most destructive diseases of sugarcane (Saccharum officinarum L.) worldwide. The pathogen spreads primarily through infected sugarcane setts and hence the use of disease-free setts is essential to prevent the disease. In order to develop immunological method for detection of C. falcatum, two proteins with molecular weights of 27 kDa and 45 kDa were purified from the mycelium of C. falcatum race Cf 05 and used as antigen source to raise polyclonal antibodies in NewZealand white rabbit. The developed polyclonal antibodies were tested for detection of C. falcatum by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The polyclonal antibodies specifically detected C. falcatum in extracts from infected plants, both in immunoblot and ELISA. The ELISA results showed that the developed polyclonal antibodies were highly specific to C.falcatum. The developed antibodies were very sensitive and could detect C.falcatum proteins even at a dilution of 1:50,000. Higher ELISA absorbance values were recorded even at an antigen dilution of 1:500. In western blot analysis, protein bands with molecular weights of 27 kDa and 45 kDa reacting to antisera raised against 27 kDa and 45 kDa mycelial proteins of C. falcatum, respectively, were detected in protein samples from red rot infected canes. The high specific reactivity and sensitivity of the antisera indicate its potential suitability for ELISA-based detection of C. falcatum.     

Resistance to Didymella applanata in red raspberry and some related species

When raspberry canes were inoculated in summer with a mycelial inoculum of Didymella applanata resistant genotypes developed relatively small lesions which produced few fruiting bodies in the following spring. Scores of the frequency of these fruiting bodies provided the best discrimination between genotypes. Very strong resistance was found in R. pileatus, R. occidentalis and R. coreanus and in hybrids of these species with red raspberry. Hybrids with R. crataegifolius were less resistant. Resistance was also found in Malling 1473/35, a derivative of red raspberry cv. Chief, and in red raspberry segregates with gene H which determines cane pubescence. Both the expression of resistance and the ranking of resistance sources for their resistance was similar for D. applanata to that reported for Botrytis cinerea.     

Delayed-type hypersensitivity response in an isogenic murine model of paracoccidioidomycosis

The specific delayed-type hypersensitivity (DTH) response was evaluated in resistant (A/SN) and susceptible (B10.A) mice intraperitoneally infected with yeasts from a virulent (Pb18) or from a non-virulent (Pb265)Paracoccidioides brasiliensis isolates. Both strains of mice were footpad challenged with homologous antigens. Pb18 infected A/SN mice developed an evident and persistent DTH response late in the course of the disease (90th day on) whereas B10.A animals mounted a discrete and ephemeral DTH response at the 14th day post-infection. A/SN mice infected with Pb265 developed cellular immune responses whereas B10.A mice were almost always anergic. Histological analysis of the footpads of infected mice at 48 hours after challenge showed a mixed infiltrate consisting of predominantly mononuclear cells. Previous infection of resistant and susceptible mice with Pb18 did not alter their DTH responses against heterologous unrelated antigens (sheep red blood cells and dinitrofluorobenzene) indicating that the observed cellular anergy was antigen-specific. When fungal related antigens (candidin and histoplasmin) were tested in resistant mice, absence of cross-reactivity was noted. Thus, specific DTH responses againstP. brasiliensis depend on both the host's genetically determined resistance and the virulence of the fungal isolate.Abbreviations DTH delayed-type hypersensitivity - DNFB dinitrofluorobenzene - FN18 Fava Netto's antigen obtained from isolate Pb18 - FN265 Fava Netto's antigen obtained from isolate Pb265 - SRBC sheep red blood cells     

Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.     

Somaclonal variants resistant to sugarcane mosaic virus and their agronomic characterization

Summary The sugarcane mosaic virus is a pathogen that causes severe disease to sugarcane. New varieties resistant to insects and pathogens have been developed in the last 70 yr through sugarcane breeding programs, but this takes between 10 and 15 yr. Tissue culture techniques are used as an aid for sugarcane improvement to increase desirable agronomic characteristics, such as disease resistance. In the present work, we report the generation of somaclonal variants from sugarcane (Saccharum spp.) cultivar PR62258 susceptible to Sugarcane Mosaic Virus, by somatic embryogenesis. These new variants identified as AT626 and BT627 are resistant to Sugarcanes Mosaic Virus strains A and B, respectively. We established an indirect enzyme linked inmunosorbent assay (ELISA) to test the presence of the viral particles in plants, and its was demonstrated that the leaves of resistant somaclones do not contain viral particles. The field performance of the somaclones AT626 and BT627 was similar to the field performance of the mother plant PR62258.     

Resistance to six viruses in the legume goat's rue {Galega orientalis Lam.)

Plants from 2nd to 6th year leys of the legume goat's rue (Galega orientalis Lam.) were tested for infection with bean yellow mosaic (BYMV), bean common mosaic (BCMV), alfalfa mosaic (AMV), broad bean stain (BBSV), red clover mottle (RCMV) and cucumber mosaic (CMV) viruses by enzyme-linked immunosorbent assay (ELISA), electron microscopy, and by sap-inoculation to various test plant species. No virus infections were observed in goat's rue in the field. Glasshouse-grown seedlings of goat's rue were inoculated with the above viruses. No virus was detected in the inoculated plants. The results suggest that goat's rue is extremely resistant to the above six viruses which are important in other forage legumes.     

Response of wheatgrasses and wheat × wheatgrass hybrids to barley yellow dwarf virus

Summary Resistance to barley yellow dwarf virus (BYDV), manifested by low enzyme-linked immunosorbent assay (ELISA) values in plants exposed to viruliferous aphids, was identified in several wheatgrasses (Agropyron spp.). ELISA results were similar for root and leaf extracts of infested plants. No difference in reaction to BYDV was found between plants grown in the field and those in the growth chamber. Interspecific hybrids were generated using pollen from single resistant plants of Agropyron spp. to pollinate soft red winter wheat spikes. Resistance in hybrids appeared to be at the level of virus replication rather than at the level of vector inoculation. The hybrids varied in their reaction to BYDV. Expression of BYDV resistance in hybrids was influenced not only by wheat genotype and Agropyron species but, in some cases, reaction varied even among hybrids between the same wheat genotype and Agropyron plant. Implications of these results are discussed.Contribution from the Purdue Univ. Agric. Exp. Stn., West Lafayette, IN 47907, and USDA-ARS. The research was supported in part by Public Varieties of Indiana. Purdue Univ. Agric. Exp. Stn. Journal No. 11656     

Diversity of Rice yellow mottle virus in Benin and Togo and Screening for Resistant Accessions

Surveys were conducted in rice fields in Benin, Cote d'Ivoire, Mali, Nigeria, Togo and Niger to assess the importance of Rice yellow mottle virus (RYMV). Diseased leaf samples were collected. In Togo, surveys were made mainly in the southern part of the country, and in Benin, all areas where rice is grown were covered. Leaf samples were serologically confirmed by ACP ELISA as bearing RYMV, propagated and all conserved. One hundred and forty‐eight (148) Beninese and 27 Togolese strains were serotyped. They were also phenotyped on three susceptible accessions including IR 64, four resistant lines with known alleles on RYMV 1 gene namely TOG 5681 (rymv 1‐3), TOG 5672 (rymv 1‐4 and RYMV 2), TOG 5674 (rymv 1.5) and Gigante (rymv 1‐2). RYMV spots with 9–100% incidences were identified. Serotyping by triple antibody sandwich (TAS) ELISA indicated that two main groups S1 and S2 coexisted in Benin with S1 being prevalent. In Togo, 26 strains were S1 and only one was S2. Phenotyping of the 148 Beninese and 27 Togolese indicated that they all attacked the three susceptible accessions, while TOG 5681 (rymv 1‐3), TOG 5672 (rymv 1‐4 and RYMV 2), TOG 5674 (rymv 1‐5) and Gigante (rymv 1‐2) remained symptomless. The use of the genes/alleles above in these countries against the disease is discussed. Three representative Beninese strains were selected to screen 48 accessions for disease resistance. Nine accessions were as susceptible as IR 64 to all strains while six (NERICA 9, NERICA 12, NERICA 13, TOG 7291, WAB56‐50, CG 14 and Moroberekan) were very resistant. Susceptibility of the six ARICA and resistance of some NERICA lines could be explained by the fact that one or both parental lines were, respectively, susceptible or resistant to the strains.     

Detection of Sap-Transmissible Viruses by ELISA in Tissue-Propagated Plants of Red Raspberry during in vitro Culture

Apple mosaic virus and raspberry bushy dwarf virus were detected by ELISA in plantlets of red raspberry still growing in vitro. The plantlets were derived from explants which were excised from plants infected by either of the viruses mentioned. Detection by ELISA of prune dwarf virus in 4-month-old in vitro cultures of sour cherry was reported earlier. Thus, application of ELISA to tissue cultured plants in vitro seems to be an appropriate method for early detection of virus-infected plant cultures.     

Malaria and hereditary ovalocytosis

Summary Hereditary ovalocytosis in Papua New Guinea is restricted to areas of endemic malaria and may confer increased resistance to the disease. The incidence of malaria was investigated in 1616 Melanesians of known red cell morphology and severity of infection determined in a smaller subsample. Ovalocytics tended to be more resistant to severe malarial infections than normocytics. The ratio of parasitaemia in 112 ovalocytics compared with 741 normocytic children was 1.05 for P. falciparum; 0.90 for P.vivax; 0.54 for P. malariae, and 0.91 for infection with any species. The difficulties in conclusively demonstrating any selective advantage of the condition are discussed.     

Spread of herbicide‐resistant weedy rice (red rice,Oryza sativa L.) after 5 years of Clearfield rice cultivation in Italy

The weedy relative of cultivated rice, red rice, can invade and severely infest rice fields, as reported by rice farmers throughout the world. Because of its close genetic relationship to commercial rice, red rice has proven difficult to control. Clearfield (Cl) varieties, which are resistant to the inhibiting herbicides in the chemical group AHAS (acetohydroxyacid synthase), provide a highly efficient opportunity to control red rice infestations. In order to reduce the risk of herbicide resistance spreading from cultivated rice to red rice, stewardship guidelines are regularly released. In Italy, the cultivation of Cl cultivars started in 2006. In 2010, surveillance of the possible escape of herbicide resistance was carried out; 168 red rice plants were sampled in 16 fields from six locations containing Cl and traditional cultivars. A first subsample of 119 plants was analysed after herbicide treatment and the resistance was found in 62 plants. Of these 119 plants, 78 plants were randomly selected and analysed at the level of the AHAS gene to search for the Cl mutation determining the resistant genotype: the Cl mutation was present in all the resistant plants. Nuclear and chloroplast microsatellite markers revealed a high correlation between genetic similarity and herbicide resistance. The results clearly show that Cl herbicide‐resistant red rice plants are present in the field, having genetic relationships with the Cl variety. Finding plants homozygous for the mutation suggests that the crossing event occurred relatively recently and that these plants are in the F₂ or later generations. These observations raise the possibility that Cl red rice is already within the cultivated rice seed supply.     

Screening for Barley yellow dwarf virus-Resistant Barley Genotypes by Assessment of Virus Content in Inoculated Seedlings

The content of Barley yellow dwarf virus (BYDV) in roots and leaves of barley seedling plants differing in their level of resistance was assessed by quantitative ELISA 1–42 days after inoculation with the strain of BYDV (PAV). High virus accumulation in roots and low concentration in leaves was characteristic of the period 9–15 days after inoculation. In leaves, the differences in virus content between resistant and susceptible genotypes became significant after 15 days and resistance to virus accumulation was better expressed 30–39 days after inoculation. Roots of resistant materials exhibited evident retardation of virus accumulation and the greatest difference in virus content between resistant and susceptible plants was detected 9 days after inoculation. By these criteria, the selected winter and spring barley cultivars and lines (in total 44 materials) fell in to five groups according to field reactions and the presence or absence of the Yd2 resistance gene. There were highly significant and positive relations between ELISA values and 5‐year field data on symptomatic reactions and grain‐yield reductions due to infection. Using the described method, resistant and moderately resistant genotypes (both Yd2 and non‐Yd2) were significantly differentiated from susceptible genotypes. The possible use of this method in screening for BYDV resistance is discussed.     

Pathogenicity of Colletotrichum gloeosporioides (Penz.) Sacc. causal agent of anthracnose in different varieties of eggplant (Solanum melongena L.) determined by levels of cross-reactive antigens shared by host and pathogen

Pathogenicity test of the fungal pathogen Colletotrichum gloeosporioides (Penz.) Sacc., causal agent for anthracnose in eggplant (Solanum melongena L.), was performed in 28 commercially cultivated eggplant varieties by analysing the antigenic patterns of host and pathogen. Through initial screening following detached leaf inoculation technique and whole plant inoculation technique, Pusa purple long (Ppl) variety was found to be the most susceptible while Shamala variety (Shav) was the most resistant. Cross-reactive antigens (CRA) shared by susceptible varieties and C. gloeosporioides was detected by immunodiffusion and immunoelectrophoresis and indirect enzyme-linked immunosorbent assay (ELISA). Such antigens could not be detected between the resistant varieties and the pathogen and also between a nonpathogen (Alternaria porri) and all the test varieties. However, ELISA showed that low levels of common antigens were present between all combinations. The level of CRA was found to decrease with increasing resistance. Indirect immunogold labeling followed by silver enhancement revealed that CRA were concentrated mainly in the cell wall regions throughout the tissue. The level of CRA was found to correlate to the pathogenicity of C. gloeosporioides in different eggplant varieties. ELISA may therefore be used to screen the commercially cultivated eggplant varieties for resistance to C. gloeosporioides.