Showing papers in "European Journal of Plant Pathology in 1997"

Induced resistance in plants and the role of pathogenesis-related proteins

Abstract: The nature of induced resistance Resistance, according to Agrios (1988) is the ability of an organism to exclude or overcome, completely or in some degree, the effect of a pathogen or other damaging factor Disease resistance in plants is manifested by limited symptoms, reflecting the inability of the pathogen to grow or multiply and spread, and often takes the form of a hypersensitive reaction (HR), in which the pathogen remains confined to necrotic lesions near the site of infection Induced resistance is the phenomenonthat a plant, once appropriately stimulated, exhibits an enhanced resistance upon 'challenge' inoculation with a pathogen Although induced resistance has been attracting attention recently (Ryals et al, 1994; Hammerschmidt and Kuc, 1995), the first systematic enquiry into induced resistance was made by Ross (1961a,b) He observed that the inducible resistance response to tobacco mosaic virus (TMV) in N gene-containing, hypersensitively reacting tobacco was not confined to the immediate vicinity of the resulting local necrotic lesions, but extended to other plant parts A ring of tissue around the developing lesions became fully refractory to subsequent infection (localized acquired resistance; Ross, 1961a), whereas challenge inoculation of distant tissues resulted in much smaller, and occasionally fewer, lesions (systemic acquired resistance (SAR); Ross, 1961b) than in non-induced plants Even leaves thatweremere initials at the time of the primary inoculation became induced, suggesting that as a result of the initial infection, a signal was generated, transported and propagated, that primed the plant to respond more effectively to subsequent infection (Bozarth and Ross, 1964) Treatments that influenced lesion size after primary infection had similar effects on lesions developing upon challenge inoculation (Ross, 1966), leading to the conclusion that the mechanisms responsible for resistance expression were the same under both conditions Only upon challenge inoculation, defense mechanisms appeared to be expressed earlier and to a greater extent (De Laat and Van Loon, 1983; Dean and Kuc, 1987)

Fungi associated with esca disease in grapevine

Abstract: Cross sections of woody stems of 309 diseased grapevines collected in France showed two kinds of necrosis typical of esca: a) A central light-colored necrosis of soft consistency, consisting of three zones, preceded by a centrally discolored wood, and b) a sectorial light-colored necrosis composed of two zones preceded by a sectorial brown necrosis. Isolations showed that different microflora was associated with each necrosis. Phaeoacremonium aleophilum and Phaeoacremonium chlamydosporum occurred in the discolored wood and the zones bordering the central decayed wood. Eutypa lata was the main fungus isolated from sectorial brown necrosis and the zones adjoining the decay wood. Phellinus punctatus was isolated from the sectorial and central decayed wood. Stereum hirsutum was present in decayed wood of 15 grapevines with esca symptoms not inhabited by P. punctatus. Wood decay tests and pathogenicity tests showed that S. hirsutum and P. punctatus were responsible for the decayed wood. Phaeoacremonium chlamydosporum and S. hirsutum produced a centrally discolored wood similar to that found in esca-affected vines. Phaeoacremonium aleophilum caused a sectorial brown necrosis of soft texture. From these studies, it was found that esca is a complex disease involving several microorganisms whose role in the process leading to wood degradation is discussed.

Recent advances in the molecular genetics of plant cell wall-degrading enzymes produced by plant pathogenic fungi

Abstract: Both saprophytic and plant parasitic fungi produce extracellular enzymes which can degrade the cell wall components of plants. These fungi not only digest plant cell wall polymers to obtain an important nutrient source but also degrade the cell wall to aid in penetrating cells and spreading through plant tissue. DeBary (1886) was the first to suggest that extracellular enzymes may be involved in the infection process of plant pathogenic fungi. Since then, much research has been focused on trying to determine the role and importance of extracellular cell wall-degrading enzymes (CWDE) to the virulence of plant pathogenic fungi. Traditionally this has been done by purifying and characterizing CWDE and examining the effect of purified or partially purified enzymes on plant cells. Despite considerable progress, this has not resulted in any definitive conclusions on the importance of CWDE to plant pathogenic fungi. However, in recent years, a new approach using recombinant DNA techniques has been employed to try to provide more conclusive evidence concerning the role of CWDE in plant pathogenesis. This review will examine recent developments in the molecular biology of CWDE of plant pathogenic fungi. Of the numerous CWDE produced by plant pathogenic fungi, most research has concentrated on the pectin degrading enzymes. This is because the pectinases are typically produced first, in the largest amounts, and are the only CWDE capable of macerating plant tissue and killing plant cells on their own (Cooper, 1983). The pectin matrix of plants is found throughout the primary cell wall but is most concentrated in the middle lamella between cells (Carpita and Gibeaut, 1993). The pectin matrix is thought to stabilize cellulose microfibrils, other neutral sugar polymers and proteins in the primary cell wall (Carpita and Gibeaut, 1993). The pectin matrix consists of homogalacturonan and rhamnogalacturonan with various degrees of methyl esterification of the carboxyl group of the galacturonate residues (McNeil et al., 1984). Both polymers have side chains of arabinans, xylans and/or arabinogalactans (McNeil et al., 1984). Fungi produce different types of pectinases that are classified by their substrates, type of lysis and mode of action on the pectin polymer. Unesterified pectate polymers can be degraded by polygalacturonase (PG), which uses hydrolytic cleavage, and pectate lyase (PL), which uses -elimination cleavage and the formation of a double bond in one of the resulting galacturonate residues (Rexová-Benková and Markovič, 1976). Esterified pectin polymers are attacked by pectin lyase (PNL) or polymethylgalacturonase (Rexová-Benková and Markovič, 1976). Rhamnogalacturonase (RHG) cleaves the bond between the alternating galactose and rhamnose residues in rhamnogalacturonan (Suykerbuyk et al., 1995). Two types of pectinases have been differentiated by their cleavage pattern: an endo form

Detection and differentiation of grapevine yellows phytoplasmas belonging to the elm yellows group and to the stolbur subgroup by PCR amplification of non-ribosomal DNA

Abstract: Primer pairs were designed from a cloned DNA probe of a strain of flavescence doree (FD) phytoplasma and from a cloned DNA probe of a strain of stolbur phytoplasma. Among an array of reference phytoplasma strains maintained in periwinkle, pair FD9f/r amplified a 1.3 kb DNA fragment only with phytoplasma strains of elm yellows (EY) group, i.e. two strains of FD and two strains of EY. Tru9I restriction analysis of the fragment amplified by FD9f/r revealed a diversity among EY-group phytoplasmas. The FD strains differed from the strains isolated from elm. The profile of the phytoplasmas infecting the grapevine samples from Catalonia and most of the samples from Northern Italy were identical to that of a FD strain. Three other profiles were detected in grapevine from Palatinate, in Germany.

Aggressiveness and other factors relating to displacement of populations of Phytophthora infestans in England and Wales

Abstract: Mating type, in vitro sensitivity to the phenylamide fungicide metalaxyl, and mitochondrial (mtDNA) haplotype were determined in some or all of 618 isolates of Phytophthora infestans from the years between 1978 and 1995. A2 mating type occurred infrequently in most but not all years and insensitivity to metalaxyl increased over time. After 1982, the mtDNA Ib haplotype was largely replaced (except for one isolate in 1986 and one in 1995) by two new haplotypes, Ia and IIa. Type Ia was much more common than type IIa. Approximately one quarter of these isolates (165) were compared using two components of fitness associated with aggressiveness (infection frequency × number of sporangia per lesion) on detached leaves of cultivars Maris Piper, Cara and Stirling, which were chosen as exhibiting increasing levels of race non-specific resistance. Isolates were compared with three ‘standard’ isolates of low, intermediate or high aggressiveness, and the data standardised for comparison between experiments. On cvs. Cara and Stirling, but not on Maris Piper, mtDNA Ia and IIa haplotypes were more aggressive than type Ib in several separate experiments. Similarly, metalaxyl sensitive phenotypes were more aggressive than insensitive phenotypes on Cara and Stirling but not on Maris Piper. The displacement of mtDNA type Ib by types Ia and IIa over this period may have been a result of the lower aggressiveness and lack of complete insensitivity to metalaxyl in type Ib isolates.

Detection and identification of Phytophthora fragariae Hickman by the polymerase chain reaction

Abstract: Phytophthora fragariae Hickman, which causes strawberry red stele and raspberry root rot, is a quarantine organism for which specific and sensitive detection methods are required to test the health of planting material. Sequences of the internal transcribed spacer regions of the ribosomal gene repeat (rDNA) were used to develop primers for P. fragariae in a nested Polymerase Chain Reaction (PCR). The fungus was readily detected in infected but symptomless roots by nested, but not single-round, PCR. It was also detected in infested water samples obtained from the Dutch General Inspection Service by nested PCR. Detection of PCR products was at least 10-fold more sensitive by PCR-ELISA than by conventional visualisation on agarose gels.

Nitric oxide preserves the level of chlorophyll in potato leaves infected by Phytophthora infestans

Abstract: Nitric oxide (NO) is a bioactive molecule involved in many physiological processes Among its biological function, NO has been proved to be cytotoxic against microorganisms in cells of the immune response, thus preventing infection We have specifically studied the effect of a NO donor, sodium nitroprusside (SNP), on the chlorophyll content in potato leaves infected with the pathogenic fungus Phytophthora infestans (Pi) Fifteen days after infection, chlorophyll content strongly decayed in water-treated potato leaf sections SNP was able to partially revert that loss in a dose-dependent manner, being the effective SNP concentrations between 10 µM and 100 µM NaNO2 and NaNO3, the SNP-derived residual products, were unable to prevent the chlorophyll loss Treatments with SNP did not affect the survival of Pi and the fungus was able to grow in a V8-agar medium containing 100 µM SNP Both the amount and the extent of germination of Pi sporangia resulted similar in the absence and in the presence of SNP Respiratory inhibitors of the cyanide-sensitive and cyanide-resistant pathways, 2,4-dinitrophenol and salicylhydroxamic acid respectively, did not change the chlorophyll levels in infected potato leaves, suggesting that NO effect should not be on mitochondrial respiration These results indicate that NO could be a protective molecule, either preserving the chloroplast membrane of infected leaf sections against the toxicity of reactive oxygen species or being directly involved in any step of the chlorophyll metabolic pathway

Selection of biological control agents for controlling soil and seed-borne diseases in the field

Abstract: Different screening methods for selection of biological control agents (BCAs), for controlling soil and seed-borne diseases, are discussed. The shortcomings of laboratory methods focused on mechanism of action are discussed and we conclude that these methods should be used with caution if candidates with multifactorial or plant mediated mechanisms of control are to be obtained. In vitro screens may be useful for specific groups of microorganisms, thus, screens for antibiotics may be relevant for Streptomyces spp., and promising results have been obtained using soil plating or precolonized agar methods to screen for mycoparasitism and competitive saprophytic ability. Experience with screening in the Nordic programme ‘Biological control of seed borne diseases in cereals’ is summarized. Research in the four participating countries – Finland, Sweden, Norway and Denmark – followed the same paradigm: that of obtaining antagonists, well adapted to different Nordic environments, and developing them as effective BCAs. Potential antagonists were isolated from different sources and in planta screening methods were developed in order to optimize selection of antagonists effective against a range of seed borne pathogens. Screens in the laboratory or greenhouse were followed by screening in the field. The different screening procedures are compared and evaluated.

Characterisation of the European pathogen population of Magnaporthe grisea by DNA fingerprinting and pathotype analysis

Abstract: The genetic variability among 41 isolates of the blast pathogen (Magnaporthe grisea) from five European rice growing countries was studied. The genealogy of the isolates was investigated by DNA fingerprinting and the results compared to the degree of similarity for (a)virulence factors. Fingerprinting grouped the isolates into five discrete lineages, that typically showed less than 65% band similarity. Within each lineage, two or more haplotypes were detected with a band similarity of 80% or higher. Each lineage showed a characteristic virulence pattern. All isolates of lineage ‘E5’ belonged to the same pathotype. The other lineages were composed of clusters of closely related pathotypes that showed variation for virulence to cultivars with certain known resistance genes, while remaining invariably (a)virulent to others. In most cases, lineage classification of an isolate could be easily inferred by its pathotype. Certain resistance genes and certain lineage-excluding resistance gene combinations appear to provide protection against all of the virulence factors sampled.

Effect of oxygen concentration on plant growth, lipidperoxidation, and receptivity of tomato roots to Pythium F under hydroponic conditions

Abstract: The effects of the nutrient solution oxygenation on the growth of tomato plants and colonization of plant roots by Pythium F707, an isolate with filamentous non-inflated sporangia, were investigated under hydroponic conditions. Lipoperoxidation was also estimated determining lipoxygenase activity and conjugated dienes. Tomato plants were grown under either a high (11-14%; Air treatment), a moderate (5.8-7%; Control) or a low (0.8-1.5%; Nitrogen treatment) oxygen concentration and inoculated or not with the pathogen. The high oxygen treatment resulted in a marked increase in plant growth, as measured by shoot and root weights. Root and top weights were about the same in the nitrogen-treated plants and the controls. In these treatments, plants started showing typical symptoms of root decay and infection within 6 days after inoculation with Pythium F, while highly oxygenated plants remained healthy throughout the experiment and showed a significant decrease in root colonization by the pathogen, as estimated by the immunoenzymatic staining procedure and isolation of thalles on selective medium. Nitrogen-treated plants and controls produced higher amounts of conjugated dienes and revealed increased lipoxygenase activities in comparison with highly oxygenated plants. These differences were more pronounced after inoculation with the pathogen. Our data suggest that increases in lipoxygenase activity detected in the present study in tomato roots grown under oxygen stress and inoculated with Pythium F may lead to degradation and disorganization of membrane lipids. That disorganization may facilitate root colonization by the pathogen and appearance of decay.

Endo-1,3- β -glucanase and cellulase from Trichoderma harzianum: purification and partial characterization, induction of and biological activity against plant pathogenic Pythium spp.

Abstract: There were indications that endo-1,3-β-glucanase (1,3-(1,3;1,4)-β-D-Glucan 3(4)-glucanohydrolase (EC 3.2.1.6)) and cellulase (1,4-(1,3;1,4)-β-D-Glucan 4-glucanohydrolase (EC 3.2.1.4)) activity of Trichoderma harzianum Rifai isolate T3 were induced in sphagnum peat moss cultivations and dual culture experiments by the presence of Pythium ultimum. Further, P. ultimum stimulated the germination of Trichoderma conidia. Endo-1,3-β-glucanase and cellulase were purified from T. harzianum isolate T3, known to control Pythium damping-off of cucumber seedlings. The enzymes were purified from the culture filtrate of the fungus by gel filtration and isoelectric focusing. The purified endo-1,3-β-glucanase was a small protein with a molecular mass of 17 kilodaltons and a pI of 5.0. Two cellulases were purified to homogeneity and had molecular masses of 40 and 45 kilodaltons respectively, and pI's of 6.4 and 7.6 respectively. Germination of encysted zoospores and elongation of germ tubes of a plant pathogenic Pythium isolate were inhibited by low concentrations of the purified enzymes. A strong synergistic effect was observed on the inhibition of cyst germination by a combination of the endo-1,3-β-glucanase and the fungicide Fongarid. Finally, a time-course study of colonization of the rhizosphere of cucumber seedlings showed that the active fungal mycelial biomass of a GUS-transformant of T. harzianum isolate T3 increased over four weeks. Trichoderma appeared to colonize healthy roots only superficially, whereas the mucilage of the root hairs and of distal parts of wounded areas or broken parts of the roots, were extensively colonized.

Operationalizing sustainability: exploring options for environmentally friendly flower bulb production systems

Abstract: Current production systems for flower bulbs in the Netherlands employ considerable quantities of pesticides and nutrients per unit area. In 1993, an association of growers and environmentalists set out to design new farming systems that meet environmental objectives in addition to economic objectives. To support the design process, an explorative study was carried out to bring together the fragmented agronomic information and to assess agro-technical options for sustainable flower bulb production with a time horizon of 10 to 15 years. Crop and inter-crop management systems representing the agro-technical components of sustainability at the farm level, were generated with a computer model by systematically varying four system characteristics, three of which represented strategic and tactical aspects of crop protection. Subjective components, one economic and two environmental objectives and various socio-economic constraints, were identified in interaction with the stakeholders. Interactive multiple goal linear programming was used to optimize the objectives at the farm level and determine the exchange value of the economic objective in terms of the environmental objectives. Calculations were carried out for two reference farm types. The results revealed that the negative impact of environment-oriented production systems on farm gross margin is importantly mitigated by strategic choices at the farm level, such as renting land and allowing a soil health improving crop, even though of low gross margin, into the rotation. In contrast, the a priori attention of the growers was focused on improving tactical pest and nutrient management at the crop level, the effect of which on farm gross margin is constrained by the strategic choices. Sensitivity analyses highlighted the need for more insight into the ecology of soil-borne growth reducing factors and their effect on crop yield. The paper describes the approach used, reports results and discusses the usefulness of the approach for the stakeholders and for disciplinary crop protection research.

Global crop production and the efficacy of crop protection--current situation and future trends.

Abstract: Actual and potential crop losses of eight major food and cash crops have been estimated by evaluating data from literature and field experiments Total losses were calculated from yield reductions due to pathogens, animal pests and weeds on a regional, continental and global level Since 1965, worldwide production of most crops has increased considerably Simultaneously, crop losses in wheat, potatoes, barley and rice increased by 4 to 10 percent, in maize, soybean, cotton and coffee losses remained unchanged or slightly decreased The efficacy of crop protection practices was calculated as the percentage of potential losses prevented by control The efficacy is highest in cotton (55 percent), it reaches only 34 to 38 percent in the food crops rice, wheat and maize The variability among cropping areas is high: In Western Europe, 61 percent of potential crop losses is prevented, in North America and Oceania 44, in all other regions 38 percent Due to the small share of Western Europe in worldwide production of 8 percent, the efficacy of actual crop protection worldwide is only 40 percent In view of population growth and rising food demand crop production has to be increased substantially As potential loss rates often increase with attainable yields high productivity largely depends on effective crop protection management Scenarios for the production of food crops by the year 2025 in developed and in developing countries are given Recent and future developments in crop protection can contribute to establish sustainability in agriculture and to preserve natural resources However, although effective control methods have been developed for most biotic yield constraints, the use of crop protection products is regulated by economic considerations rather than by food demand

Comparison of visual head blight ratings, seed infection levels, and deoxynivalenol production for assessment of resistance in cereals inoculated with Fusarium culmorum

Abstract: Seven spring wheat, 13 barley, 14 oats and 20 winter wheat genotypes were inoculated at flowering in 1993 and 1994 with mixed conidial suspensions of 8 isolates of Fusarium culmorum. Four weeks after inoculation, head blight was recorded in the field. After harvest, seed infection was assessed by a Freezing Blotter Test in the laboratory. Seed samples were also analyzed for deoxynivalenol (DON) content. Differences were found in head blight rating, the levels of infected seeds and the DON content between wheat, barley, and oats and between cultivars. Highly significant correlations were found between the percentage of heavily infected seed and the DON content. The weighted mean value of infected seeds and DON content were also significantly correlated. No significant correlation was found between head blight rating in the field and DON content. The level of infected seeds, as determined by the Freezing Blotter Test, was a better indication of the DON content in the seeds than the head blight rating. This visual assessment of levels of infected seeds gives a reliable estimate of resistance to Fusarium.

Inhibition of egg hatch of the potato cyst nematode Globodera rostochiensis by chitinase-producing bacteria

Abstract: Plant-parasitic nematodes are major agronomic pests. Purified commercial chitinase inhibited egg hatch of the potato cyst nematode, Globodera rostochiensis (Ro1) in vitro by up to 70% when compared with an untreated control. A screening strategy was devised to isolate chitinase-producing bacteria from a soil with no documented history of damage due to potato cyst nematodes in the last 30 years and that was cropped with potato cv. ‘Kerr's Pink’. Only 137 of 3,200 bacterial isolates tested for chitinase production on chitin agar plates were chitinase-positive (i.e. about 4%). All the chitinase-producing bacteria tested in vitro could reduce the hatch of G. rostochiensis eggs, some by up to 90% compared with the controls. One of these strains, M1-12, was identified as Stenotrophomonas maltophilia and a second strain UP1 was classified as a Chromobacterium sp. based on morphological and biochemical tests. The inoculum level and the incubation time influenced the degree of inhibition of egg hatch of G. rostochiensis by M1-12 and UP1 in vitro. An initial cell density of 106 CFU ml-1 or greater and an incubation time of two weeks was needed to inhibit egg hatch. The longer UP1 was allowed to act on the eggs of G. rostochiensis the greater the level of inhibition. Strains M1-12 and UP1 also reduced the ability of G. rostochiensis to hatch in soil microcosms planted with potato seed tubers cv. ‘D'sir'e’. The inhibition of egg hatch of G. rostochiensis by chitinase-producing bacteria is suggested as a biocontrol strategy for the defence of potato crops from potato cyst nematodes.

Baseline sensitivity and cross-resistance to demethylation-inhibiting fungicides in Ontario isolates of Sclerotinia homoeocarpa

Abstract: Four hundred and thirty-five isolates of Sclerotinia homoeocarpa from eight populations in southern Ontario were tested for sensitivity to the demethylation-inhibiting (DMI) fungicides, propiconazole, myclobutanil, fenarimol and tebuconazole. The isolates were collected in summer 1994 just prior to legal DMI fungicide use on turfgrass in Ontario. There were wide variations in sensitivities, and seven of the eight populations were very sensitive to the fungicides. Based on mean EC50 and the distribution of DMI sensitivity, one population near the U.S. border was suspected of having been previously exposed to DMI fungicide. Pairwise comparisons of EC50 values for the different fungicides showed low to moderate correlations between fungicides. EC50 values of myclobutanil and propiconazole had the best correlation, followed by the pair of tebuconazole and fenarimol. Other pairwise comparisons were not statistically significant except for a barely significant relationship between EC50 values of myclobutanil and tebuconazole. For field populations of plant pathogens, cross-resistance to different DMI fungicides may not be as strong as conventionally thought. The data collected here will allow comparison to subsequent years to look for detectable shifts in S. homoeocarpa sensitivity to DMI fungicides as they become more frequently used in Ontario.

Biological control of cereal seed-borne diseases by seed bacterization with greenhouse-selected bacteria

Abstract: About 400 bacterial strains, isolated from roots of wild and cultivated plants, were screened for effects against diseases caused by Drechslera teres and/or Microdochium nivale in greenhouse tests and against common bunt caused by Tilletia caries in field tests. Four of the strains showed good biocontrol activity <70% disease reduction) against D. teres and T. caries both in screenings and field tests. One Pseudomonas isolate, MA 342, strongly and reliably suppressed both D. teres and T. caries in the field, while effects against M. nivale were weaker. The effects could not be enhanced by varying pre-application or seed application procedures. This isolate could be stored as a suspension in a refrigerator, frozen or applied to seeds for at least one month without loosing its disease controlling ability.

Changes in the structure of Arabidopsis thaliana roots induced during development of males of the plant parasitic nematode Heterodera schachtii

Abstract: Plant parasitic nematodes of the genus Heterodera show a high degree of sexual dimorphism, which is reflected by different nutritional demands and differences in the structure of the induced specific syncytial feeding site in the plant. The determination of the sex of the nematode Heterodera schachtii and other related species was repeatedly reported to be dependent on trophic factors, which are provided by the induced syncytia. The structural differences of syncytia induced by H. schachtii in roots of Arabidopsis thaliana were analysed at the anatomical and ultrastructural level. Syncytia of males were induced in the root pericycle. The developing syncytium then expanded into procambial or cambial cells of the vascular cylinder. Differentiated vascular elements were not included. The expansion of the syncytium triggered the proliferation of cambial and peridermal tissues, in a manner similar to secondary growth, and the formation of additional xylem and phloem elements. In comparison to syncytia associated with females, syncytia associated with males were less hypertrophied and were composed of more cells. Distinct cell wall openings were mostly found between the few strongly hypertrophied syncytial elements at the actual feeding site in the pericycle. The ultrastructure was very similar to female-associated syncytia but showed conspicuous differences in the structure and localization of cell wall ingrowths. These ingrowths were rare and weakly developed and occurred not only at the interface with xylem elements but also at the internal and external walls of the syncytia. After feeding had ceased at the end of the third developmental stage the syncytia degenerated.

Characterization and classification of phytoplasmas from wild and cultivated plants by RFLP and sequence analysis of ribosomal DNA

Abstract: Restriction fragment length polymorphism and sequence analysis of PCR-amplified ribosomal DNA were used to identify and classify phytoplasmas associated with diseases of various wild and cultivated plants. The diseases examined were either not known before or the presumable causal agents were not yet identified and characterized or were only known from other geographic areas. New diseases examined were those causing virescence and phyllody of Bunias orientalis and Cardaria draba. Both were associated with strains of the aster yellows phytoplasma. The same type of aster yellows phytoplasma was also found to be associated with yellows and phyllody diseases of Portulaca oleracea, Stellaria media, Daucus carota ssp. sativus, and Cyclamen persicum. In German and French DNA samples from diseased Trifolium repens, the clover phyllody phytoplasma was identified, which could clearly be distinguished from other phytoplasmas of the aster yellows group. Strains of the stolbur phytoplasma were detected in big bud-affected tomatoes and almost exclusively in Convolvulus arvensis. In Cirsium arvense and Picris echioides two distinct phytoplasmas were identified which showed relationship to the sugarcane white leaf phytoplasma group but may represent a new group or subgroup. In Conyza (syn.: Erigeron) canadensis a phytoplasma of the X-disease group was detected. A strain from Gossypium hirsutum showed the same restriction profiles as the faba bean phyllody phytoplasma.

Production and characterization of a monoclonal antibody specific to the M serotype of plum pox potyvirus

Abstract: A monoclonal antibody to an Albanian isolate of plum pox potyvirus (PPV) was obtained (MAbAL), that specifically recognized strain M of this virus. The specificity of MAbAL, assessed by comparative ELISA on 130 PPV isolates of different geographical origin, 22 of which were also tested by comparative IC-PCR, gave consistent and highly reproducible results. MAbAL seems to be elicited by a stable surface determinant that makes it particularly suitable for successful use under a wide range of conditions. MAbAL is an useful addition to the panel of PPV-specific MAbs available to date.

Some characteristics of the transmission of grapevine leafroll associated virus 3 by Planococcus citri Risso

Abstract: Some characteristics of the acquisition and transmission of GLRaV-3 by Planococcus citri were determined by ELISA testing and transmission experiments. Groups of five insects were used, i.e. the advisable minimum group size suggested by the results of ELISA of insect groups of various sizes. The virus was transmitted to only 1/10 test plants each of which had been exposed to a group of insects fed on GLRaV-3 infected plants for at least three days, eventhough more than 80% of the insect groups were expected to contain viruliferous individuals under these conditions. Viruliferous mealybugs transferred to potato plants could retain the virus for up to 24 h, but lost the capacity for effective transmission to vines within 1 h after transfer. In newly infected vines, the virus remained latent or undetectable by ELISA for at least 13 months.

Genetic diversity in Fusarium oxysporum f.sp. dianthi and Fusarium redolens f.sp. dianthi

Abstract: Pathogenic isolates were selected representing all known vegetative compatibility groups (VCGs) and races of Fusarium oxysporum sensu lato from Dianthus spp. On basis of differences in the internal transcribed spacer region of the ribosomal DNA, six VCGs were classified as F. oxysporum f.sp. dianthi and four as F. redolens f.sp. dianthi. All VCGs of F. oxysporum f.sp. dianthi were characterized by unique restriction fragment length polymorphisms (RFLPs), unique overall esterase profiles, and unique virulence spectra, supporting a clonal lineage concept. Two VCGs of F. oxysporum f.sp. dianthi nevertheless comprised more than one race, but races within the same VCG shared the same distinct overall virulence spectrum. VCGs belonging to F. redolens f.sp. dianthi also had unique RFLPs and unique virulence spectra, but had grossly identical esterase profiles. Three new races (9, 10 and 11) are described for F. oxysporum f.sp. dianthi, and four for F. redolens f.sp. dianthi. Two races previously considered lost were recovered; race 7 was identified as a member of VCG 0021 of F. oxysporum f.sp. dianthi while race 3 was identified as a distinct VCG and race of F. redolens f.sp. dianthi. A summary of races and VCGs in F. oxysporum f.sp. dianthi and F. redolens f.sp. dianthi is presented.

A foliar spray of micronutrient solutions induces local and systemic protection against powdery mildew (Sphaerotheca fuliginia) in cucumber plants

Abstract: A single spray of solutions of 0.005M H3BO3, 0.0025M CuSO4, and 0.0025 MnCl2, on the upper surface of the first true leaf of cucumber plants 2 h before inoculation with a conidial suspension of Sphaerotheca fuliginea, induced systemic protection against powdery mildew in leaves 2 and 3 without causing any damage on the induced leaf (first leaf). A similar level of systemic protection was observed when plants were induced by micronutrients, 2, 24 and 72 h before challenge with S. fuliginea. The level of protection induced by various concentrations varied from solution to solution. In general, the systemic protection induced by K2HPO4 was similar to that by the microelements. Spraying of a 1:1 mixture of phosphate and micronutrient solutions did not improve the systemic protection over that obtained with each of the solutions alone. Increasing the inoculum concentration of S. fuliginea increased the number of powdery mildew colonies produced on both induced and non-induced plants and has relatively affected the systemic protection on induced plants. A single foliar spray of micronutrient solutions, as a prophylactic treatment, on the upper surface of all the leaves of 3-leaf stage cucumber plants significantly inhibited powdery mildew development. A single spray of MnCl2 on leaf 1 elevated peroxidase activity in the soluble fraction and caused an enhancement of β-1,3-glucanase content in the ionically bound fractions of leaf 2 of non-inoculated plants. Forty-eight hours after inoculation, the level of both fractions of the enzymes increased in non-treated plants and decreased (β-1,3-glucanase) or remained unchanged (peroxidase) in treated (induced) plants as compared to non-treated plants. The possible mechanism for this protection, and the use of microelements and phosphate solutions as inducers for systemic protection and as agents for disease control are discussed.

Coat protein-mediated resistance to isolates of barley yellow dwarf in oats and barley.

Abstract: Tissue cultures of GAF30/Park oats were biolistically co-transformed with constructs containing the coat protein (CP) genes of the P-PAV, MAV-PS1 or NY-RPV isolates of barley yellow dwarf virus (BYDV), together with a construct containing the bar gene for herbicide resistance and the uidA reporter gene. Transformed, herbicide-resistant tissue cultures were screened by PCR for the presence of the CP genes. Fertile regenerated plants were recovered from some CP-transformed tissue cultures. T1 progeny of these plants were screened for resistance to the BYDV isolate corresponding to the introduced gene by inoculation with viruliferous aphids followed by ELISA tests. Variation in ELISA values for GAF30/Park control plants made interpretation of the data difficult, but oat plants resistant to each of the three isolates of BYDV (ELISA values less than 0.3; virus titers equivalent to less than 25% of infected controls) were identified in T1 generations. Further testing of MAV-PS1 CP-transformed lines to the T2 generation, NY-RPV CP-transformed lines to the T3 generation and P-PAV CP-transformed lines to the T4 generation identified further resistant plants. Similarly, immature embryos and calli of the barley cultivar Golden Promise were biolistically bombarded with constructs containing the CP gene of the P-PAV isolate of BYDV and the bar and uidA reporter genes, lines of self-fertile P-PAV CP-transformed barley plants were developed, and T1plants were screened for resistance to P-PAV. Eight plants from six lines showed moderate to high levels of resistance to P-PAV that correlated with the presence of the CP gene. Plants giving low ELISA values were also found in other lines, even though the CP gene was not detected in these plants. Some T2 plants derived from resistant parents that contained the CP gene were themselves highly resistant.

Early vascular defense reactions of cotton roots infected with a defoliating mutant strain of Verticillium dahliae

Abstract: Susceptible and resistant cotton lines were cytologically and histochemically investigated for their defense reactions to a highly aggressive and defoliating strain of Verticillium dahliae, a fungus responsible for vascular wilt. Cytochemistry showed that early responses consisted of reinforcement in structural barriers with polysaccharides, including callose and cellulose. Ultrastructural modifications of parenchyma cells of the vascular tissues were associated with strong production of terpenoids and phenolics. These defense reactions were detected early in roots of the resistant line, one to four days after inoculation, while they were seen later in roots of the susceptible line.

Virulence variation and DNA polymorphism in Sphaerotheca fuliginea, causal agent of powdery mildew of cucurbits

Abstract: Strains of Sphaerotheca fuliginea, one of the causal agents of powdery mildew of cucurbits, were examined for differences in virulence, mating type and DNA polymorphism. The 28 strains were chosen to be diverse according to host and geographic origin. Characterization of virulence phenotypes was based on the expression of symptoms on 4 species of cucurbits and 6 cultivars of melon. Two pathotypes, capable of attacking either cucumber cv. ‘Marketer’ and melon cv. ‘IranH’ and squash cv. ‘Diamant’ or cucumber cv. ‘Marketer’ and melon cv. ‘IranH’ were observed. Tests on melon cultivars revealed 3 races. In tests of sexual compatibility with reference strains, heterothallism was observed for all isolates. Frequency of the two mating types differed significantly in the population. DNA polymorphism was determined both by restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers (ITS) and 5.8S DNA amplified by the polymerase chain reaction and by random amplified polymorphic DNA (RAPD). For any one of the 11 restriction enzymes tested all strains presented an identical pattern of ITS RFLP. RAPD analysis, using 22 primers which provided reproducible patterns, revealed a relatively low degree of polymorphism. Furthermore, cluster analysis based on RAPD data (152 markers) did not separate groups within the species S. fuliginea. No association could be found between virulence, mating type, geographical and host origin and RAPD patterns. The lack of association between phenotypic and molecular markers and the close fit to linkage equilibrium for the characters examined suggest that recombination may play a role in populations of S. fuliginea.

Characterization of Rhizoctonia solani AG 2 isolates causing bare patch in field grown tulips in the Netherlands

Abstract: During a spring survey in 1991, 130 isolates of R. solani were collected in 25 commercial flower bulb fields from diseased plants occurring in bare patches. On the basis of hyphal fusion frequency and pathogenicity to flower bulbs, tulip isolates were provisionally assigned to AG 2-t to distinguish these isolates from AG 2-1 isolates which were non-pathogenic to bulbs. Hyphal fusion frequency of a subgroup of 7 AG 2-t isolates was highly variable when paired with 7 AG 2-1 isolates (2-75%), thus making assignment of AG 2-t isolates to AG 2-1 inconclusive. The mean hyphal fusion frequency among AG 2-t isolates was 65% (±6%) indicating AG 2-t to be a relatively homogeneous group. Hyphal fusion frequency among AG 2-1 isolates was highly variable with a mean 51% (±25%) indicating AG 2-1 to be a heterogeneous group. The optimum growth temperature for AG 2-t and AG 2-1 isolates on malt peptone agar was 20-25 °C. The host range of AG 2-t and two AG 2-1 isolates comprised tulip, iris, hyacinth and lily at both 9 and 18 °C, and cruciferous, sugarbeet and lettuce seedlings at 18 °C. Six other AG 2-1 isolates were pathogenic to cruciferous seedlings, but not to any of the bulbous crops. The tested narcissus, Tagetes patula, tomato, potato, wheat, leek and maize cultivars were not susceptible to AG 2-t and AG 2-1 isolates. Statistical analysis using a proportional-odds model revealed significant differences in aggressiveness between R. solani AG 2-t isolates and differences in susceptibility between tulip and iris cultivars. At 18 °C, but not at 9 °C, isolates representing AG 2-2, AG 4, AG 5 and AG BI were pathogenic to bulbous crops. In addition to bare patch causing AG 2-t isolates, other anastomosis groups may cause disease in field grown tulips. For the development of optimal crop rotation schedules, the impact of bulb rot causing isolates under field conditions needs further study.

Identification of Piper yellow mottle virus, a mealybug-transmitted badnavirus infecting Piper spp. in Southeast Asia

Abstract: A previously undescribed badnavirus was found to be a causal agent of a disease of black pepper (Piper nigrum) in Malaysia, the Philippines, Sri Lanka and Thailand, and was also associated with a disease of betelvine (P. betle) in Thailand. Disease symptoms included chlorotic mottling, chlorosis, vein-clearing, leaf distortion, reduced plant vigor and poor fruit set. The virus, named Piper yellow mottle virus (PYMV), had non-enveloped bacilliform virions averaging 30 × 125 nm in size and containing a double-stranded DNA genome. An isolate of PYMV from Thailand was transmitted by mechanical inoculation and by the citrus mealybug, Planococcus citri, from infected P. nigrum and P. betle to healthy P. nigrum seedlings, which developed symptoms similar to those observed in naturally-infected plants. A serological relationship between PYMV and isolates of banana streak (BSV) and sugarcane bacilliform (ScBV) viruses, but not six other badnaviruses, was detected by immunosorbent electron microscopy (ISEM). Genomic PYMV sequences were amplified by polymerase chain reaction (PCR) using badnavirus-specific oligonucleotide primers, and sequence analysis comparisons of the putative reverse transcriptase (RT) domain showed PYMV to be closely related to other mealybug-transmitted badnaviruses. Black pepper infected with PYMV sometimes contained one or more isometric virus-like particles, and PYMV may therefore be only one component of a virus complex infecting black pepper in Southeast Asia.

Subdivision and genetic structure of four populations of Venturia inaequalis in Switzerland

Abstract: Analyses of four populations of Venturia inaequalis in Switzerland were performed to obtain information about migration and to predict the probable speed of the spread of new pathotypes able to overcome resistance, e.g. Vf-resistance, of new cultivars. Genetic and haplotype diversity was calculated based on allele frequencies of random amplified polymorphic DNA (RAPD) markers and the internal transcribed spacer (ITS)-region of ribosomal DNA, which are regarded to be neutral, and the β-tubulin locus which may be under selection pressure. Within-population diversity was found to be quite similar over all four populations. Normalised haplotype diversity based on RAPD and ITS data was very high with a mean of 0.95. Diversity among populations (GST) was consistent over all neutral loci with a low mean of 0.04, but reached the high value of 0.26 for the selected β-tubulin locus. Low GST based on neutral loci may suggest a high level of gene flow. Considering these results, new pathotypes would be expected soon outside their place of identification. But actual gene flow is easily overestimated because of effects of gene flow in the past. However, naturally occurring gene flow could be increased by human activity. Therefore, it is very difficult to predict durability of the Vf-resitance in Switzerland.

A simulation model for the development of brown rust epidemics in winter wheat

Abstract: A model simulating the progress of Puccinia recondita severity, expressed as a percentage of rusted leaf area (both as average and its 95% confidence interval) on individual wheat leaves over the course of a growing season, with a time step of one day, was elaborated using laboratory and field data from literature. Data on the stages of each infection cycle (uredospore germination, penetration, latency, uredium eruption and infectiousness) were transformed into model parameters by curve fitting, Montecarlo stochastic procedures, corrections and empirical assumptions. Data on host growth, like the timing of all phenological stages, the dynamic of the green area of each leaf from appearance to complete senescence, and tillering were obtained from a specific sub-model. Model validation was performed on actual data not used in model building and representing a wide range of conditions (several winter wheat cultivars grown at eight locations in northern Italy between 1990 and 1994) by using subjective, non-parametric and parametric tests: it revealed a satisfactory agreement between the data simulated by the model and actual data.