Selection of natural isolates of Trichoderma spp. for biocontrol of Polymyxa betae as a vector of virus causing rhizomania in sugar beet

The soil fungus Polymyxa betae, Keskin, besides being a root parasite, plays a role of a vector in dissemination of Beet necrotic yellow vein virus (BNYVV) causing rhizomania in sugar beet. An alternative to its chemical control is the application of antagonistic microorganisms suppressing proliferation of the fungal vector. In the present work, 66 Trichoderma isolates have been obtained from sugar beet plantations from diverse locations in Slovakia. The ability of the selected isolates to grow at low temperature (10 °C) and to suppress the colonization of roots with P. betae and the multiplication of BNYVV in roots under glasshouse conditions were tested. The roots of sugar beet seedlings growing in the BNYVV-infested soil were analyzed by serological ELISA test using monoclonal and polyclonal antibodies for the presence of BNYVV and checked microscopically for the occurrence of cystosori of P. betae. The efficacy of the selected strains to suppress the proliferation of BNYVV varied on the average between 21 and 68%. On the basis of these tests, candidate strains for practical application in biocontrol of sugar beet rhizomania were selected.     

Superiority of a soil debris isolation method over a beet seed colonization method for assay of Rhizoctonia solani at high soil inoculum densities.

A quantitative soil debris isolation method (all debris from known weight of soil plated) and a garden beet seed saprophytic colonization method were compared over a 1-year period for assaying Rhizoctonia solani population. Four fields of different soil textures were selected. Within each field four areas of healthy and four areas of diseases (rhizoctonia root and crown rot) sugarbeets were sampled bimonthly from August 1976 until June 1977. The maximum numbers of R. solani colonies obtained by the debris method were 2 per gram of soil in areas of healthy beets, and 11 per gram of soil in areas of diseased sugarbeets. At such high inoculum densities the beet seed colonization method underestimated R. solani populations, because the inoculum per unit of soil exceeded the numbers of beet seeds per unit of soil available for colonization. Modifications of the beet seed method did not significantly alter results of colonization assays. Ranked correlation comparisons of assay methods yielded r = 0.81 for all data.     

Effect of phenanthrene and Rhodotorula glutinis on arbuscular mycorrhizal fungus colonization of maize roots

The effect of the polycyclic aromatic hydrocarbon (PAH) phenanthrene and the yeast Rhodotorula glutinis on the arbuscular mycorrhizal fungus (AMF) Glomus geosporum colonizing maize roots, was studied. During a 90-day experiment, the highest G. geosporum colonization values were found in control plants. Mycorrhiza root length, measured both on the basis of percentage of root colonization and on the activity of succinate dehydrogenase, showed similar patterns in different phenanthrene treatments. The presence of phenanthrene in the substrate reduced G. geosporum intraradical colonization. The presence of R. glutinis did not enhance AMF colonization in the presence of phenanthrene. The biomass of the external mycelium estimated on the basis of the fatty acid 16:1 omega 5 concentration showed a progressive increase through time, and the amounts of this fatty acid differed among treated and untreated substrates. However, this increase was found to be lowest in the phenanthrene and Rhodotorula treatment at 60 days. There was less phenanthrene accumulation in roots of maize inoculated with AMF and the yeast than in roots inoculated only with AMF. A similar pattern was observed in the phenanthrene content of G. geosporum spores collected after 90 days.     

Soil suppressiveness to Rhizoctonia solani and microbial diversity

Rhizoctonia solani anastomosis group 2-2IIIB causes damping-off, black root rot and crown rot in sugar beet (Beta vulgaris). Based on experiences of growers and field experiments, soils can become suppressive to R. solani. The fungus may be present in the soil, but the plant does not show symptoms. Understanding the mechanisms causing soil suppressiveness to R. solani is essential for the development of environmentally friendly control strategies of rhizoctonia root rot in sugar beet. A bioassay that discriminates soils in their level of disease suppressiveness was developed. Results of bioassays were in accordance with field observations. Preliminary results indicate an active role of microbial communities. Our research is focused on the disentanglement of biological mechanisms causing soil suppressiveness to R. solani in sugar beet. Therefore, we are handling a multidisciplinary approach through experimental fields, bioassays, several in vitro techniques and molecular techniques (PCR-DGGE).     

Nuclear magnetic resonance: a tool for imaging belowground damage caused by Heterodera schachtii and Rhizoctonia solani on sugar beet

Belowground symptoms of sugar beet caused by the beet cyst nematode (BCN) Heterodera schachtii include the development of compensatory secondary roots and beet deformity, which, thus far, could only be assessed by destructively removing the entire root systems from the soil. Similarly, the symptoms of Rhizoctonia crown and root rot (RCRR) caused by infections of the soil-borne basidiomycete Rhizoctonia solani require the same invasive approach for identification. Here nuclear magnetic resonance imaging (MRI) was used for the non-invasive detection of belowground symptoms caused by BCN and/or RCRR on sugar beet. Excessive lateral root development and beet deformation of plants infected by BCN was obvious 28 days after inoculation (dai) on MRI images when compared with non-infected plants. Three-dimensional images recorded at 56 dai showed BCN cysts attached to the roots in the soil. RCRR was visualized by a lower intensity of the MRI signal at sites where rotting occurred. The disease complex of both organisms together resulted in RCRR development at the site of nematode penetration. Damage analysis of sugar beet plants inoculated with both pathogens indicated a synergistic relationship, which may result from direct and indirect interactions. Nuclear MRI of plants may provide valuable, new insight into the development of pathogens infecting plants below- and aboveground because of its non-destructive nature and the sufficiently high spatial resolution of the method.     

Occurrence and diversity of yeasts involved in fermentation of West African cocoa beans

Samples of cocoa beans were taken on two separate occasions during heap and tray fermentations in Ghana, West Africa. In total 496 yeast isolates were identified by conventional microbiological analyses and by amplification of their ITS1-5.8S rDNA-ITS2 regions. For important species the identifications were confirmed by sequencing of the D1/D2 domain of the 5' end of the large subunit (26S) rDNA. Assimilations of organic acids and other carbon compounds were conducted. For dominant yeasts intraspecies variations were examined by determination of chromosome length polymorphism (CLP) using pulsed-field gel electrophoresis. For the heap fermentations maximum yeast cell counts of 9.1 x 10(7) were reached, whereas maximum yeast counts of 6.0 x 10(6) were reached for the tray fermentations. Candida krusei was found to be the dominant species during heap fermentation, followed by P. membranifaciens, P. kluyveri, Hanseniaspora guilliermondii and Trichosporon asahii, whereas Saccharomyces cerevisiae and P. membranifaciens were found to be the dominant species during tray fermentation followed by low numbers of C. krusei, P. kluyveri, H. guilliermondii and some yeast species of minor importance. For isolates within all dominant species CLP was evident, indicating that several different strains are involved in the fermentations. Isolates of C. krusei, P. membranifaciens, H. guilliermondii, T. asahii and Rhodotorula glutinis could be found on the surface of the cocoa pods and in some cases on the production equipment, whereas the origin of e.g. S. cerevisiae was not indicated by the results obtained. In conclusion, the results obtained show that fermentation of cocoa beans is a very inhomogeneous process with great variations in both yeast counts and species composition. The variations seem to depend especially on the processing procedure, but also the season and the post-harvest storage are likely to influence the yeast counts and the species composition.     

Suppression of damping-off disease in host plants by the rhizoplane bacterium Lysobacter sp. strain SB-K88 is linked to plant colonization and antibiosis against soilborne Peronosporomycetes

We previously demonstrated that xanthobaccin A from the rhizoplane bacterium Lysobacter sp. strain SB-K88 suppresses damping-off disease caused by Pythium sp. in sugar beet. In this study we focused on modes of Lysobacter sp. strain SB-K88 root colonization and antibiosis of the bacterium against Aphanomyces cochlioides, a pathogen of damping-off disease. Scanning electron microscopic analysis of 2-week-old sugar beet seedlings from seeds previously inoculated with SB-K88 revealed dense colonization on the root surfaces and a characteristic perpendicular pattern of Lysobacter colonization possibly generated via development of polar, brush-like fimbriae. In colonized regions a semitransparent film apparently enveloping the root and microcolonies were observed on the root surface. This Lysobacter strain also efficiently colonized the roots of several plants, including spinach, tomato, Arabidopsis thaliana, and Amaranthus gangeticus. Plants grown from both sugar beet and spinach seeds that were previously treated with Lysobacter sp. strain SB-K88 displayed significant resistance to the damping-off disease triggered by A. cochlioides. Interestingly, zoospores of A. cochlioides became immotile within 1 min after exposure to a SB-K88 cell suspension, a cell-free supernatant of SB-K88, or pure xanthobaccin A (MIC, 0.01 microg/ml). In all cases, lysis followed within 30 min in the presence of the inhibiting factor(s). Our data indicate that Lysobacter sp. strain SB-K88 has a direct inhibitory effect on A. cochlioides, suppressing damping-off disease. Furthermore, this inhibitory effect of Lysobacter sp. strain SB-K88 is likely due to a combination of antibiosis and characteristic biofilm formation at the rhizoplane of the host plant.     

Insect feeding-induced differential expression of <Emphasis Type="Italic">Beta vulgaris</Emphasis> root genes and their regulation by defense-associated signals

Root responses to insect pests are an area of plant defense research that lacks much information. We have identified more than 150 sugar beet root ESTs enriched for genes responding to sugar beet root maggot feeding from both moderately resistant, F1016, and susceptible, F1010, genotypes using suppressive subtractive hybridization. The largest number of identified F1016 genes grouped into the defense/stress response (28%) and secondary metabolism (10%) categories with a polyphenol oxidase gene, from F1016, identified most often from the subtractive libraries. The differential expression of the root ESTs was confirmed with RT-PCR. The ESTs were further characterized using macroarray-generated expression profiles from F1016 sugar beet roots following mechanical wounding and treatment of roots with the signaling molecules methyl jasmonate, salicylic acid and ethylene. Of the examined root ESTs, 20, 17 and 11% were regulated by methyl jasmonate, salicylic acid and ethylene, respectively, suggesting these signaling pathways are involved in sugar beet root defense responses to insects. Identification of these sugar beet root ESTs provides knowledge in the field of plant root defense and will lead to the development of novel control strategies for control of the sugar beet root maggot.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users     

Taproot promoters cause tissue specific gene expression within the storage root of sugar beet

The storage root (taproot) of sugar beet (Beta vulgaris L.) originates from hypocotyl and primary root and contains many different tissues such as central xylem, primary and secondary cambium, secondary xylem and phloem, and parenchyma. It was the aim of this work to characterize the promoters of three taproot-expressed genes with respect to their tissue specificity. To investigate this, promoters for the genes Tlp, His1-r, and Mll were cloned from sugar beet, linked to reporter genes and transformed into sugar beet and tobacco. Reporter gene expression analysis in transgenic sugar beet plants revealed that all three promoters are active in the storage root. Expression in storage root tissues is either restricted to the vascular zone (Tlp, His1-r) or is observed in the whole organ (Mll). The Mll gene is highly organ specific throughout different developmental stages of the sugar beet. In tobacco, the Tlp and Mll promoters drive reporter gene expression preferentially in hypocotyl and roots. The properties of the Mll promoter may be advantageous for the modification of sucrose metabolism in storage roots.     

SUGAR BEET YELLOWS: A PRELIMINARY STUDY OF THE DISTRIBUTION AND INTERRELATIONSHIPS OF VIRUSES AND VIRUS STRAINS FOUND IN EAST ANGLIA, 1955-57

Aphid transmissions to sugar beet seedlings from yellowed sugar beet leaves collected from commercial fields in East Anglia during the summers of 1955, 1956 and 1957, showed the occurrence of two yellowing viruses. One was sugar beet yellows virus (SBYV) and produced vein-etch and yellowing symptoms on beet seedlings in the glasshouse; the other produced yellowing but no etch. These two viruses were apparently unrelated, so that sugar beet tolerant to one of them would not necessarily be tolerant to the other. The second virus, called 'sugar beet mild yellowing virus' (SBMYV), decreased the root yield of sugar beet plants grown under glass, by as much as did the milder SBYV strains, but less than did the severe SBYV strains. The proportions of the two viruses in the samples differed from year to year and from place to place.     

In vitro attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa

The ability of yeasts to attach to hyphae or conidia of phytopathogenic fungi has been speculated to contribute to biocontrol activity on plant surfaces. Attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa was determined using in vitro attachment assays. Yeasts were incubated for 2 d on potato dextrose agar (PDA) prior to experimentation. A total of 292 yeasts cultured on PDA were screened for their ability to attach to conidia of B. cinerea; 260 isolates (89.1%) attached to conidia forming large aggregates of cells, and 22 isolates (7.5%) weakly attached to conidia with 1 or 2 yeast cells attached to a few conidia. Ten yeasts (3.4%), including 8 isolates of Cryptococcus laurentii, 1 isolate of Cryptococcus flavescens, and an unidentified species of Cryptococcus, failed to attach to conidia. All non-attaching yeasts produced copious extracellular polysaccharide (EPS) on PDA. Seventeen yeast isolates did not attach to hyphal fragments of B. cinerea, R. solani, and S. homoeocarpa after a 1 h incubation, but attachment was observed after 24 h. Culture medium, but not culture age, significantly affected the attachment of yeast cells to conidia of B. cinerea. The 10 yeast isolates that did not attach to conidia when grown on agar did attach to conidia (20%-57% of conidia with attached yeast cells) when cultured in liquid medium. Attachment of the biocontrol yeast Rhodotorula glutinis PM4 to conidia of B. cinerea was significantly greater at 1 x 10(7) yeast cells x mL(-1) than at lower concentrations of yeast cells. The ability of yeast cells to attach to fungal conidia or hyphae appears to be a common phenotype among phylloplane yeasts.     

BNYVV-derived dsRNA confers resistance to rhizomania disease of sugar beet as evidenced by a novel transgenic hairy root approach

Agrobacterium rhizogenes-transformed sugar beet hairy roots, expressing dsRNA from the Beet necrotic yellow vein virus replicase gene, were used as a novel approach to assess the efficacy of three intron-hairpin constructs at conferring resistance to rhizomania disease. Genetically engineered roots were similar in morphology to wild type roots but were characterized by a profound abundancy, rapid growth rate and, in some cases, plagiotropic development. Upon challenge inoculation, seedlings showed a considerable delay in symptom development compared to untransformed or vector-transformed seedlings, expressing dsRNA from an unrelated source. The transgenic root system of almost all seedlings contained no or very low virus titer while the non-transformed aerial parts of the same plants were found infected, leading to the conclusion that the hairy roots studied were effectively protected against the virus. This readily applicable novel method forms a plausible approach to preliminarily evaluate transgenic rhizomania resistance before proceeding in transformation and whole plant regeneration of sugar beet, a tedious and time consuming process for such a recalcitrant crop species.     

Effects of sugar beet chitinase IV on root-associated fungal community of transgenic silver birch in a field trial

Heterogenous chitinases have been introduced in many plant species with the aim to increase the resistance of plants to fungal diseases. We studied the effects of the heterologous expression of sugar beet chitinase IV on the intensity of ectomycorrhizal (ECM) colonization and the structure of fungal communities in the field trial of 15 transgenic and 8 wild-type silver birch (Betula pendula Roth) genotypes. Fungal sequences were separated in denaturing gradient gel electrophoresis and identified by sequencing the ITS1 region to reveal the operational taxonomic units. ECM colonization was less intense in 7 out of 15 transgenic lines than in the corresponding non-transgenic control plants, but the slight decrease in overall ECM colonization in transgenic lines could not be related to sugar beet chitinase IV expression or total endochitinase activity. One transgenic line showing fairly weak sugar beet chitinase IV expression without significantly increased total endochitinase activity differed significantly from the non-transgenic controls in the structure of fungal community. Five sequences belonging to three different fungal genera (Hebeloma, Inocybe, Laccaria) were indicative of wild-type genotypes, and one sequence (Lactarius) indicated one transgenic line. In cluster analysis, the non-transgenic control grouped together with the transgenic lines indicating that genotype was a more important factor determining the structure of fungal communities than the transgenic status of the plants. With the tested birch lines, no clear evidence for the effect of the heterologous expression of sugar beet chitinase IV on ECM colonization or the structure of fungal community was found.     

THE VALUE OF GLASSHOUSE TESTS WITH SEEDLINGS IN SELECTING PLANTS TOLERANT TO BEET YELLOWS VIRUS

Commercial strains of sugar beet, and breeders' lines of Beta vulgaris and B. vulgaris subsp. maritima , were exposed to infection with SBY and SBYN strains of beet yellows virus, using varying numbers of Myzus persicae as vectors, so as to cause a wide range of symptoms. The severity of the symptoms was assessed by scoring for superficial veinal necrosis ('etch'). All varieties of sugar beet tested were susceptible to infection, but the severity of the symptoms varied, particularly between the cultivated and wild beet types.
The scores for severity of symptoms made in the glasshouse were positively correlated with similar scores made in a field experiment using two cultivated and two wild beet types. The symptom scores were also positively correlated with losses in root and sugar yields caused by the virus.     

Diagnosis and Molecular Variability of an Argentinean Population of Nacobbus aberrans with Some Observations on Histopathology in Tomato

Diagnosis of an Argentinean population of Nacobbus sp. infecting sweet pepper (lamuyo) was carried out including morphology, scanning electron microscopy, and molecular studies. In light of our morphometric, molecular and host-range results, we consider the studied population to belong to N. aberrans s. l., and by host range tests the population is assigned to the "sugar beet group." ITS-PCR analysis on individual male and immature female specimens of this population yielded amplification products of approximately 922 bp. RFLP profiles and sequencing of the ITS region revealed that, in addition to the host group, the present population can be assigned to the "Argentina 2" group. Disease development and histopathology were investigated with glasshouse observations using tomato, pepper, sugar beet and potato seedlings exposed to nematode infection for 45 days at 28 ± 2°C. Histopathology of tomato roots confirmed that all immature stages and young females and males are migratory, whereas mature females are obligate sedentary endoparasites. Rather than syncytia, large regions of cortical necrosis and cavities were detected in tomato swellings infected by juveniles. However, syncytia were associated only with adult females. Large root galls, hyperplasia, abnormal proliferation of lateral roots and asymmetry of root structure were common anatomical changes induced by the nematode feeding in tomato roots.     

Manganese deficiency in sugar beet and the incorporation of manganese in the coating of pelleted seed

Summary A laboratory study, three glasshouse tests and eight field experiments on commercial farms in East Anglia during 1972 to 1974 tested the effect of incorporating manganese in the coating of pelleted seed on the manganese nutrition and yield of sugar beet. The pelleting material readily absorbed manganese from solution but most of the manganese was held in plant-available forms. Tests in the glasshouse showed that manganese sulphate or manganous oxide were likely to be the most effective seed-pellet additives, but manganese sulphate was harmful to sugar beet in slightly acid potting compost.Incorporating manganous oxide in the pellet prevented early symptoms of deficiency on sugar beet in field experiments and replaced an early foliar spray but the plants developed manganese deficiency later. The treatment was an economic and effective method of supplying manganese to sugar-beet seedlings too small to spray but, should manganese deficiency be prolonged, the plants will need spraying when large enough.     

Microbial colonization in the seagrass Posidonia spp. roots

The pattern of colonization by microorganisms on root surfaces from three species of seagrass belonging to the genus Posidonia was assessed. Microbial abundance on roots was measured by two electronic microscope techniques. Trends in microbial colonization between species and root order were defined. In addition, eutrophication status of the sampling sites and physiological status of Posidonia oceanica (L.) Delile roots have been taken into account. Our results show high microbial abundance in the Mediterranean species P. oceanica, in comparison with the low rates of colonization found in the Australian species P. australis Hook f. and P. sinuosa Cambridge et Kuo. Microbial density tended to decrease as root order increased, and living roots always showed higher microbial abundance than dead ones. Colonization of P. oceanica roots at the three sites with different environmental status follows different trends according to root order. It is suggested that root age influences the rate of microbial colonization of seagrass roots and that colonization of root surface by microorganisms is associated with organic exudates from the roots rather than with decaying root tissues.     

Study of the aggressiveness of Rhizoctonia solani isolates

This paper presents an in vitro test to screen the pathogenicity of different Rhizoctonia solani isolates on a host range. The level of aggressivity of the different isolates was different for several host plants tested. There were significant differences between the crops and the isolates tested. In general, the disease level was higher on beans, lettuce and cabbage. In carrot and rye grass the level of infection was lower for the isolates of R. solani tested. The potato isolates of R. solani were less aggressive than the isolates coming from maize, fodder beet and sugar beet. The R. solani isolates were also biochemically characterized by pectic zymograms: the isolates Rs0401 (from maize) and Rs0504 (from sugar beet) belong both to the anastomosis group AG2-2.     

The influence of soil-applied fungicides and previous cropping on the development of violet root rot of sugar beet

The proportion of sugar-beet roots infected by Helicobasidium purpureum increased most rapidly in September and October. Violet root rot was not controlled by fungicides applied at drilling or in July. Heavily infected roots yielded 31% less sugar than healthy or lightly infected roots. Sugar beet following infected carrots lifted or ploughed in during July had no more violet root rot than when following barley or fallow, but the beet crop became heavily infected when it followed carrots left in the ground until December, whether they were then lifted or ploughed in. Eight varieties of sno-ar beet did nnt differ in siiscenrihilitv to violet root rot.