ALBERT

Notes: A new continuous cell line (GF-1) was established and characterized. The GF-1 cell line, derived from the fin tissue of a grouper, Epinephelus coioides (Hamilton), was maintained in L15 medium containing 5% foetal bovine serum (FBS) at 28 °C, and has been subcultured more than 160 times since 1995. The majority of GF-1 cells are fibroblast-like, together with some epithelioid cells. Spontaneous transformation of GF-1 cells occurred during subculture 50 to subculture 80, and led to an increase of plating efficiency, less requirement of FBS and de novo susceptibility to grouper nervous necrosis virus (GNNV). Cytopathic effects (CPEs) could be observed in GF-1 cells 3–5 days post-infection with pancreatic necrosis virus (IPNV), hard clam reovirus (HCRV), eel herpes virus Formosa (EHVF) and GNNV. In addition, abundant GNNV particles were found in the cytoplasm of GNNV-infected GF-1 cells using electron microscopy and nucleic acids of GNNV virus were detected by polymerase chain reaction in the culture medium of GNNV-infected cells after CPE appeared. The experimental results indicated that GF-1 can effectively proliferate fish nodavirus and is a promising tool for studying fish nodavirus.

Notes: Protective immunity against enteric septicaemia of catfish (ESC) following immunization with Edwardsiella ictaluri bacterins and exposure to live E. ictaluri was investigated. Mean cumulative percentage survival was significantly higher (P 〈inlineGraphic alt="leqslant R: less-than-or-eq, slant" extraInfo="nonStandardEntity" href="urn:x-wiley:01407775:JFD310:les" location="les.gif"/〉 0.05) in controlled live vaccinates (100%) than in immersion and oral bacterin vaccinates (68.3% and 50.0%, respectively). Bactericidal activity against E. ictaluri by peritoneal macrophages from controlled live vaccinates (85.9%) was significantly greater (P 〈inlineGraphic alt="leqslant R: less-than-or-eq, slant" extraInfo="nonStandardEntity" href="urn:x-wiley:01407775:JFD310:les" location="les.gif"/〉 0.05) than bactericidal activity of macrophages from immersion bacterin vaccinates (71.4%) or non-vaccinates (68.1%). No significant (P 〉 0.05) difference was found in the bactericidal activity of macrophages from oral bacterin vaccinates and macrophages from controlled live vaccinates. The E. ictaluri-specific antibody response of controlled live (0.08 OD) and immersion bacterin vaccinates (0.11 OD) was significantly higher (P 〈inlineGraphic alt="leqslant R: less-than-or-eq, slant" extraInfo="nonStandardEntity" href="urn:x-wiley:01407775:JFD310:les" location="les.gif"/〉0.05) than that of oral bacterin vaccinates and non-vaccinates (0.01 OD) 15 days post-vaccination. A significantly higher antibody response was seen in controlled live vaccinates (0.17 OD), when compared to other vaccinates or non-vaccinates 33 days after vaccination. Neither immersion nor oral bacterins protected the vaccinates against ESC. Controlled live E. ictaluri immunization of channel catfish resulted in production of specific antibodies, increased macrophage bactericidal activity and protection against ESC.

Notes: A non-virulent Carnobacterium sp., designated strain K1, isolated from the gastrointestinal tract of Atlantic salmon, Salmo salar L., which produced inhibitory substances against bacterial fish pathogens, was examined in vitro for characteristics important for the colonization of the fish gastrointestinal tract and in vivo for persistence in the tract after oral dosing. In vitro growth experiments showed that the cells of this strain were metabolically active in both the intestinal mucus and faeces from salmonids. The production of growth inhibitors against the two common fish pathogens Vibrio anguillarum and Aeromonas salmonicida by Carnobacterium sp. strain K1 was demonstrated in vitro in mucus and faecal extracts. Furthermore, it was demonstrated that the Carnobacterium cells remained viable in the gastrointestinal tract for several days and that no detrimental effect to the fish was observed as a result of the presence of the bacterium.

Notes: A vital staining technique with fluorescein diacetate (FDA) and propidium iodide (PI) was used for determination of viability of myxosporean stage spores of Myxobolus artus and actinosporean stage spores of M. cultus. Viable spores stained green with FDA and non-viable spores stained red with PI were clearly distinguishable in M. artus myxosporean spores released from live infected fish, while the spores collected from pseudocysts were not well stained with FDA. Immediately after release from the oligochaete, Branchiura sowerbyi, actinosporean spores of M. cultus, stained bright green in the sporoplasms and red in the spore processes. In vitro survivability tests revealed that the longevity of M. artus spores was 5 months at 25°C and 15 months at 18°C. Drying and ultraviolet irradiation (36 000 mW s cm–2 for M. artus myxosporean spores, 600 mW s cm–2 for M. cultus actinosporean spores) were most effective as sporicidal treatments.

Notes: Six monoclonal antibodies (mabs) produced against Pasteurella piscicida whole cells, cultured under iron limitation conditions, were characterized and specificity tested. Two of the mabs (WE15H1D7 and WE3D6C11) appeared to be specific for P. piscicida lipopolysaccharide, while mabs WE14C10F9 and WE12D7D8 crossreacted with Photobacterium species. The remaining two mabs, WE9D6D8 and VP4B11, crossreacted with other bacterial genera. All mabs reacted with material present in the extracellular products of the pathogen as well as to whole cells, suggesting that the antigens to which antibodies are directed slough off the cell membranes or are secreted to the culture medium. The potential of the mabs as tools for the diagnosis of pasteurellosis is discussed.

Notes: The uptake, metabolism, tissue distribution and excretion of four sulphonamides and trimethoprim following bath treatment of Atlantic halibut, Hippoglossus hippoglossus L., were studied. Bath treatment using a concentration of 200 μg ml–1 for 72 h resulted in peak sulphadimidine concentrations in muscle and abdominal organ homogenates of 32·6 and 68·2 μg g–1, respectively. The corresponding values were 24·4 and 73·4 μg g–1 for sulphaguanidine, 6·1 and 45·1 μg g–1 for sulphamethoxazole, 2·1 and 15·1 μg g–1 for sulphadimethoxine, and 99 and 169 μg g–1 for trimethoprim. After a 72-h treatment, approximately 90% of the sulphadimethoxine and sulphamethoxazole present in tissues was found as the N4-acetylated metabolite, whereas for sulphadimidine and sulphaguanidine, the N4-acetylations were from 9 to 23%. Based on these preliminary absorption studies, sulphadimidine was chosen as the companion sulphonamide to trimethoprim. Using a combination of 500 μg ml–1 sulphadimidine and 100 μg ml–1 trimethoprim in the bath for 72 h, peak muscle and liver concentrations of 262 and 312 μg g–1, respectively, for sulphadimidine and 32·8 and 83·6 μg g–1, respectively, for trimethoprim were achieved. Elimination half-lives (t1/2β) for sulphadimidine were calculated to be 35 and 48 h for muscle and liver, respectively. The corresponding values for trimethoprim were 98 and 116 h. Using the 95% confidence limit for single observations (95% prediction limit) and a maximum residue limit (MRL) value of 0·05 μg g–1 for trimethoprim and 0·1 μg g–1 for sulphadimidine, the elimination times (Et95) for muscle and liver were calculated to be 18 and 26 days, respectively, for sulphadimidine and 40 and 55 days, respectively, for trimethoprim. Minimum inhibitory concentrations (MIC) values against selected strains of Vibrio sp. were equal to or above 128 μg ml–1 for sulphadimidine, between 0·25 and 4·00 μg ml–1 for trimethoprim and between 0·4 and 8·8 μg ml–1 for various ratios of the sulphadimidine:trimethoprim combination. In the tested ratios, the combined antimicrobial action of trimethoprim and sulphadimidine were synergistic, as revealed by their fractional inhibitory concentration (FIC) indices. In general, the 1:5 trimethoprim sulphadimidine ratio showed the highest degree of synergism. Using a combination of 500 μg ml–1 sulphadimidine and 100 μg ml–1 trimethoprim in the bath for 72 h, concentrations greater than a MIC value of 0·8 μg ml–1 were maintained for 22 days in muscle and 29 days in liver. In a laboratory challenge experiment using Vibrio anguillarum strain HI 11347, a significantly lower mortality was observed in the drug-treated group (40%) compared to the untreated control group (93%).

Notes: The effect of iron limitation, using the iron-chelating agent 2,2 dipyridyl, on the electrophoretic profiles of outer membrane proteins (OMPs) and extracellular products (ECPs) from 21 Pasteurella piscicida strains isolated from Europe and Japan was investigated. In addition, the effect of iron-limited and iron-surplus growth conditions on caseinase activity in culture supernatants of the pathogen was examined. The majority of P. piscicida strains, from Greece, Italy and France, cultured under iron-limited conditions, produced four novel OMPs (63 and three at and above 200 kDa). In contrast, iron-regulated outer membrane proteins were not induced in Japanese strains. Electrophoretic analysis of the ECPs from the pathogen grown under iron surplus and iron limitation revealed a large range of products and additional high molecular mass (MM) bands were evident under iron-limited conditions. When culture supernatants were analysed for their activity, most of the bacteria tested showed elevated activities under iron limited conditions. Finally, neither hydroxamate nor phenolate type siderophores could be detected with any of the chemical assays used.

Notes: Infectious salmon anaemia (ISA) is a viral disease of farmed Atlantic salmon, Salmo salar L., in Norway. However, in laboratory experiments, the virus has also been found to propagate in trout, Salmo trutta L., where it is a persistent infection. It is crucial for the management of ISA in Norway, and for the prevention of spread to fish in other countries, that possible carriers of the virus are found. Another possible salmonid reservoir species is the rainbow trout, Oncorhynchus mykiss (Walbaum). After experimental infection, the ISA virus was found to be present in O. mykiss as late 28 days after infection, with a peak around day 20. The infection resulted in a significant drop in the haematocrit and haemorrhages on the liver in some specimens. The ISA virus was seen budding from endothelial cells lining blood vessels in the heart ventricle of challenged rainbow trout. It is concluded that the ISA virus is able to propagate in O. mykiss and that this species may function as a reservoir when cultured in areas where the virus is found. The virus does not seem to cause any significant mortalities of infected rainbow trout.

Notes: An efficient clearance and degradation system may be needed during microbial invasion which otherwise would lead to severe inflammation and eventually death. Non-specific defence mechanisms in fish play an important role at all stages in infection. The non-specific humoral defence including proteases, lysins and agglutinins, for example, in mucosal secretion is the first line of defence, whereas mucosal lining cells function as the second barrier against invasion. Blood cells, especially granulocytes and monocytes, may destroy microbes present in the circulation and may function as the third line of defence. Finally, endocytically active cells such as endothelial cells, macrophages and granulocytes in organs and tissues may take up and degrade microbes or microbial products. The endocytic and degradation processes strongly depend on the effectiveness of the reticuloendothelial system which consists of endothelial cells and macrophages that line small blood vessels (e.g. sinusoids and ellipsoids). Potentiation of non-specific defence mechanisms may occur during microbial invasion, leading to more efficient clearance and destruction of pathogens or other harmful substances. In microbial invasion, an inflammatory response such as elevated production of antimicrobial substances is often encountered. Central cells in the production of antimicrobial substances are macrophages and granulocytes, and microbial products in inflammation may alter the cells function to a more activated state in vivo. Activated cells may enhance their antimicrobial capacity and efficiency by producing higher amounts and more active antimicrobial agents. This review concerns the non-specific defence system and gives an introduction to some of the known non-specific humoral substances and their induction/suppression, and provides a more extensive introduction to cytokine research and immunomodulation. Cellular aspects of non-specific defence, including macrophages and their products, are discussed in the light of their function in the reticuloendothelial system in fish.

Notes: The variation of virulence of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD) in salmonid fish, was studied by infecting rainbow trout, Oncorhynchus mykiss (Walbaum), with two isolates (strains 325 and 932) from diseased Atlantic salmon, Salmo salar L., and one isolate (strain 4366) from an apparently healthy Atlantic salmon. Coho salmon, Oncorhynchus kisutch (Walbaum), were injected with the strain 932 to estimate difference in fish species resistance. Fish were removed by random sampling for other study purposes, a study design possible with analysis of lifetime distributions incorporating both sampling-, death- and survival-times. At the end of the experiment, the rainbow trout infected with strains 325, 932 and 4366 had a survival probability of 33%, 51% and 72%, respectively. The coho salmon infected with strain 932 had a 26% survival probability. The strain differences were significant according to the log-rank test, and the risk ratio between the strains ranged from 1·8 to 5·4. The strain from the apparently healthy fish was least virulent. The survival of the fish species was different over time. Rainbow trout were more likely to die early in the time course, but high numbers of coho died later, resulting in an overall risk of mortality of 1·4 in favour of rainbow trout. Differences in virulence may reflect changed selective pressure on R. salmoninarum when introduced from feral stocks into the environment of fish farms.

Notes: Macroscopic and microscopic features of natural and experimental Flexibacter maritimus infection, and epidemiological aspects of the disease, have been reported in a number of species of fish in Tasmanian aquaculture including Atlantic salmon, Salmo salar L., rainbow trout, Oncorhynchus mykiss (Walbaum), greenback flounder, Rhombosolea tapirina Günther, and striped trumpeter, Latris lineata (Bloch & Schneider). There is a great deal of consistency in the pathology in salmonids and non-salmonid species, with erosive lesions of external surfaces being the most prominent clinical sign. Experimentally induced disease of salmonids and flounder is similar to natural infection. Mature lesions show dermal and gill erosion, with dermal bacterial invasion into the dense connective tissue and occasionally underlying musculature, but a remarkable lack of inflammatory response. The earliest lesions show consistent fragmentation and degeneration of the epithelium, with infiltration of amorphous protein-like materials and occasionally intra-epithelial cellular inflammatory cells, plus congestion and haemorrhage of the superficial dermis, but without visible bacteria in standard sections. Variable scale loss, oedema and a low level of inflammation in scale pockets, plus variable small adherent bacterial mats, are evident before full epithelial erosion.

Notes: Adult Japanese eels, Anguilla japonica Temminck & Schlegel, (200–250 g, 45–55 cm) were immunized by intramuscular injection with goat IgG. After 5 weeks, eel immunoglobulin (Ig) was purified using affinity chromatography. The purified eel Ig was used to immunize rabbits to produce anti-eel Ig antibody. The highest antibody ELISA value in eels was reached 3 weeks after initial immunization with goat IgG, and then gradually decreased. The antibody could still be detected at 140 days post-immunization. The optimal temperature for antibody production was 30°C. Freund’s complete adjuvant and secondary immunization both increased antibody production in eels.

Notes: The molecular mechanisms involved in the invasion of Aeromonas hydrophila (strain PPD 134/91) into host cells were studied in vitro using a carp epithelial cell line. Bacterial fractions were extracted with potassium thiocyanate (KSCN) to investigate the adhesins involved. Two groups of adhesins were found. The major group was high molecular weight proteins with the largest component being a 43-kDa protein. Amino terminal sequence analysis indicated that this may have been an outer membrane porin. This supports previous suggestions that a 43-kDa outer membrane protein may be important in adhesion of a human isolate of A. hydrophila. The minor group of adhesins were low molecular weight proteins likely to be less effective in mediating bacterial adhesion and invasion into carp epithelial cells.

Notes: Mass mortalities of hatchery-reared juvenile groupers have occurred in southern Taiwan. The diseased fish swam in a darting, corkscrew fashion. Light microscopy revealed vacuolation in the brain tissue. Electron microscopy showed numerous non-enveloped, cytoplasmic viral particles (20–25 nm in diameter) in the brain cells, and many virions were enclosed in the membrane-bound organelles of the cells. Two structural proteins of the purified grouper virus, with molecular weights of 44 and 43 kDa, were revealed by SDS-PAGE. Moreover, the results of RT-PCR and nested PCR diagnosis using primers specific to the T2 and T4 target segments of striped jack nervous necrosis virus (SJNNV) RNA2 genes suggest that this virus is a fish nodavirus, and is designated as GNNV 9410 strain (grouper nervous necrosis virus strain 9410). This is the first case report of viral nervous necrosis among marine fish in Taiwan.

Notes: Purified microcystin-LR (MC-LR) was administered via the dorsal aorta to brown trout, Salmo trutta L., or rainbow trout, Oncorhynchus mykiss (Walbaum), and, within 24 h, a dose of 300 μg MC-LR kg–1 caused increased activities in the blood by enzymes originating mainly from the liver, i.e. lactate dehydrogenase (LDH) and alanine transaminase (ALT). A dose of 75 μg MC-LR kg–1 significantly increased liver enzyme activities in the blood of brown trout at 24 h, but was without effect on rainbow trout, whereas 25 μg MC-LR kg–1 had no effect on blood LDH or ALT activities in either species. However, histopathological analysis of liver from both species following administration of the lowest toxin dose showed hepatocyte swelling and necrosis. Liver damage was more severe in brown trout compared to rainbow trout following administration of 300 μg MC-LR kg–1, showing disruption of the parenchymal architecture. After 48 h, there was a dose-dependent increase in the hepatosomatic index in both species. It is concluded that brown trout are less tolerant to MC-LR than rainbow trout.

Notes: Effluents from three fish hatcheries were monitored for the discharge and subsequent downstream distribution of infectious pancreatic necrosis virus (IPNV). Samples of springwater and surface water, and tissues from salmonid and non-salmonid fish were assayed for IPNV. Water samples were processed to recover virus by adsorption to an electropositive, microporous filter matrix. No IPNV was detected in surface water collected above fish hatcheries or in hatchery springwater supplies. The virus could be detected for at least 19.3 km below the point of effluent discharge from hatcheries and the prevalence of IPNV infection in stream-resident fish was 2.8%.

Notes: The objective of this study was to determine the growth of Trypanosoma danilewskyi (Laveran & Mesnil, 1904) from goldfish, Carassius auratus (L.), in medium containing no serum, or in medium supplemented with either 10% fish serum (goldfish, carp, or tin foil barb) or 10% mammalian serum (horse or foetal bovine). After 10 days, the number of trypanosomes in flasks containing tin foil barb serum increased by nearly 700% over the initial inoculum. Similarly, a substantial increase in the number of parasites was seen after 10 days in the cultures containing carp and goldfish serum. After 6 weeks, there was more than a 15-fold increase of T. danilewskyi in cultures containing goldfish serum. Medium containing no serum or mammalian serum failed to support the growth of parasites. Therefore, serum-related factors within fish blood are required for the propagation of T. danilewskyi isolated from infected goldfish. Since T. danilewskyi can be propagated in vivo and in vitro in the presence of homologous proteins, cultured and wild-type forms can be compared to determine if cultured parasites can be used as analogues of natural life-cycle stages.

Notes: Pathogenicity and cultural experiments described here provide futher evidence that a distinct species of Aphanomyces is responsible for much of the characteristic pathology of epizootic ulceration syndrome (EUS). Zoospores from 58 fungal isolates were injected intramuscularly in snakehead fish, Channa striata (Bloch). These fungi comprised: Aphanomyces strains isolated from EUS-affected fish; saprophytic Aphanomyces, Achlya and Saprolegnia spp. from infected waters; and further saprolegniaceous fungi involved in other diseases of aquatic animals. Only the Aphanomyces strains isolated from fish affected by EUS, Australian red spot disease (already considered synonymous with EUS) or mycotic granulomatosis described from Japan were able to grow invasively through the fish muscle and produce the distinctive EUS lesions. In contrast to Aphanomyces astaci Schikora, the EUS-Aphanomyces was shown to be unable to infect noble crayfish, Astacus astacus L. The snakehead-pathogenic strains were further distinguished from all the other fungi under comparison by their characteristic temperature-growth profile and inability to grow on certain selective fungal media.

Notes: A birnavirus (infectious pancreatic necrosis virus, IPNV), three rhabdoviruses (viral haemorrhagic septicaemia virus, VHSV; infectious haematopoietic necrosis virus, IHNV; and spring viraemia of carp virus, SVCV) and an iridovirus (isolate from a sheatfish) were investigated with regard to their morphogenetic interactions with cells in culture. In cells infected with birnavirus, a granular viromatrix, single virions randomly distributed in the cytoplasm, viral particles aggregated in pseudocrystals and cytoplasmic tubuli similar in diameter to that of the virus were found. Rhabdoviruses entered the cells by viropexis and replicated within the cytoplasm. Maturation occurred predominantly at the cell membrane and sporadically at membranes of the Golgi cisternae. Inclusion bodies were found partially consisting of viral nucleocapsids. After budding, new virions were found adsorbed to the cell membrane. Viral haemorrhagic septicaemia virus, known to exhibit an atypical shape because of preparative procedures, could be identified by immunostaining using two monoclonal antibodies directed against G- and N-proteins and colloidal gold. Iridoviruses entered the cells by viropexis. Viral particles were found in coated vesicles. Subsequently, vesicles without a clathrin coat were detected. Replication occurred within prominent cytoplasmic inclusion bodies. Isometric viral nucleocapsids were transported in an unknown manner to the cell membrane and matured by budding.

Notes: A survey of external lesions on Atlantic tomcod from the St Lawrence River was carried out between mid-December 1992 and the end of January 1993 (n = 1674). Jaw ulcerations were observed in 3.8% of the fish. These ulcers were usually single, and located at the junction between the skin epidermis and oral mucosa. They progressed from shallow lesions to deep ulcers with pathological fracture of the facial bone secondary to osteitis. The aetiology of these lesions could not be determined. Fish with ulcers were generally older than those of the monitored spawning population. The reason for a higher prevalence in older tomcods is unknown, but decreased immune functions and healing capacity caused by ageing are suggested as potential predisposing factors.

Notes: Mycobacterium spp. isolated from food and ornamental fish in Thailand (TB1, TB40, TB267 and TB268), and the type strains Mycobacterium marinum (NCIMB 1298), M. fortuitum (NCIMB 1294) and M. chelonae (NCIMB 1474) were cultured in Long’s medium, Eagle’s minimum essential medium, Sauton’s medium and modified Sauton’s medium. The latter enabled excellent growth and production of extracellular products (ECP) from TB 40, TB267, TB268 and M. marinum NCIMB 1298 in particular, whereas growth and production of ECP for all strains was limited in Long’s medium. SDS-PAGE protein profiles of ECPs from 14-day culture supernatants showed major bands at 65 and 〈14 kDa. After 2 days culture at the higher temperature of 37°C (heat shock), the production of ECP from all mycobacteria strains except M. marinum averaged approximately four- to 10-fold higher than from strains cultured for 14 days at 28°C. Enzyme testing for the type strains indicated only mucinase activity for M. marinum, while lipase and RNase activities were detected for M. chelonae and M. fortuitum. Protease and DNase activities could not be detected for any of the Mycobacterium spp. tested. The medium lethal dose (LD50) of ECP to rainbow trout and Nile tilapia was greater than 400 μg protein fish–1.

Notes: Two trials were conducted to assess the effects of repeated prophylactic formalin treatments on the gill structure of salmonids. In trial 1, which involved Atlantic salmon, Salmo salar L., approaching smoltification in a commercial facility, fish were treated with either 167 or 250 mg l-1 formalin for 90 min every 2 weeks for 12 weeks. Formalin-treated salmon had slight, but not significant, increases in the frequency of lamellar fusion, numbers of lamellar mucous cells, and numbers of an endemic gill ciliate, Trichophyra piscium, after 6 and 12 weeks of treatment. In trial 2, which involved juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), fish were treated with 200 mg l-1 formalin for 60 min twice weekly for 12 weeks. Significant effects were limited to an increase in the numbers of mucous cells present on gill lamellae. In both trials, there was no evidence of lamellar oedema or necrosis of lamellar epithelial cells.

Notes: A Sphaerospora sp. (Myxosporea) infection (presumably S. truttae) was identified on a trout farm in northeastern Italy. Parasites were detected in kidneys from infected brown trout, Salmo trutta L., over a 2-year period. Extrasporogonic, sporogonic stages and mature spores were simultaneously detected in the same fish. Traditional diagnostic methods for Sphaerospora spp. rely on the detection of the myxosporean developmental stages in Giemsa-stained kidney smears or haematoxylin-eosin stained tissue sections. A histochemical method was employed where 10 biotinylated lectins (Con-A, DBA, SBA, GS-I, PHA-P, LEA, PWM, RCA1, WGA and UEA-I) and the avidin-biotin-peroxidase complex (ABC) were used on Sphaerospora-infected brown trout renal tissues and kidney imprints. Five monoclonal antibodies against PKX (Mab12, MabA3, MabC5, MabD4 and MabB4) were also tested. A lectin glycoconjugate binding pattern for Sphaerospora spp. is presented. This staining method shows that SBA lectin (Glycine max agglutinin) is a useful tool for the detection of the Sphaerospora spp. Only MabB4 bound some of the most mature sporogonic stages. In contrast Mabs12, A3, C5 and D4, and GS-I lectin (Griffonia simplicifolia agglutinin) did not stain any of the Sphaerospora spp. stages, but did bind very specifically to the sporogonic and extrasporogonic stages of PKX, the causative agent of proliferative kidney disease (PKD).

Notes: A soluble immunomodulating β(1,3)-glucan, laminaran, was given in drinking water to yolk-sac larvae of Atlantic halibut, Hippoglossus hippoglossus L. The larvae absorbed laminaran when the concentration was 25 mg l–1. After 5 days exposure to laminaran, each larva contained 2.3 ng and 46 ng of 3H- and 125I-labelled laminaran, respectively. After 10 days exposure, each larva contained 4.5 ng and 47 ng of 3H- and 125I-laminaran, respectively. The absorption was confirmed by fluorescence microscopy of larvae exposed to fluorescein-labelled laminaran (FITC-laminaran). The fluorescence was localized to cells in the intestinal epithelial layer and the skin epithelial layer.

Notes: Isolates of Tapesia yallundae and Tapesia acuformis were subjected to Random Amplified Polymorphic DNA (RAPD) assay. Amplification products common to isolates of either species were cloned and primers were generated from each sequence for use in conventional PCR. The primer pair derived from a T. yallundae specific RAPD marker amplified a product only from DNA of T. yallundae isolates and not from DNA of a range of other fungal species associated with the stem base disease complex of cereals. Similarly, the primer pair generated from a T. acuformis-specific RAPD marker amplifed a product only from DNA of T. acuformis isolates. Quantitative assays were developed for both species of Tapesia from these primer pairs, using competitive PCR. Competitive PCR was used to determine the level of colonization of seedlings by each species in glasshouse- and field-inoculated cereal hosts and results compared to those for conventional seedling disease assessment.

Notes: Sixteen chemicals from different groups of known resistance inducers were tested as a soil drench in a humosoil : sand mix infested with an isolate of Rhizoctonia solani AG-4. Among these, 5-nitrosalicylic acid (2 mM aqueous solution), o-acetylsalicylic acid (2 mM), 2,6-dichloroisonicotinic acid (0.25 mM), 2-aminoisobutyric acid (2 mM) and lichenin (2 mM) controlled pre-emergence damping-off and post-emergence seedling mortality of bean cv. Dufrix, but gave little or weak and variable disease control in cucumber cv. Delikatess. Two-component mixtures of these five chemicals controlled the disease effectively in bean, mixtures containing 2-aminoisobutyric acid being the most effective, with seedling stands of up to 94% in infested soil. As none of these five chemicals (≤ 2 mM) reduced mycelial growth of R. solani on PDA plates, induced resistance in seedling tissues is implied in their disease control. Control was augmented in both hosts when any one of the five inducers was applied in a mixture with a cell suspension (1 × 109 cells mL−1) of an antagonistic fluorescent strain of Pseudomonas fluorescens; these mixtures showed additive effects and provided much better disease control in bean than in cucumber. The free-radical scavengers (antioxidants) ascorbic acid, benzoic acid, gluconic acid lactone and thiourea inhibited R. solani growth in vitro and efficiently controlled both pre-emergence damping-off and post-emergence seedling mortality of bean; ascorbic acid and benzoic acid also offered significant protection in cucumber. However, disease control values were reduced substantially when inducers were applied with antioxidants, because of strong antagonistic interactions in most of these mixtures. Probable mechanisms of disease control by 5-nitrosalicylic acid, o-acetylsalicylic acid, 2,6-dichloroisonicotinic acid, 2-aminoisobutyric acid and lichenin and their interactions with P. fluorescens and antioxidants are discussed.

Notes: Psorosis, sometimes also associated with ringspot symptoms, is a widespread and damaging disease of citrus in many parts of the world including South America and the Mediterranean basin. We describe the application of RT-PCR and DAS-ELISA diagnostics to an isolate of citrus ringspot virus (CtRSV-4) and other virus isolates associated with this disease. Fragments of cDNA from bottom-component RNA of CtRSV-4 were cloned and sequenced, and PCR primers were designed, 5′ACAATAAGCAAGACAAC upstream, and 5′CCATGTCACTTCTATTC downstream. RT-PCR experiments using these primers allowed detection of CtRSV-4 in infected citrus leaves down to a tissue dilution of 1/12 800 representing 2 μg of tissue, and less sensitive detection of the related citrus psorosis-associated virus (CPsAV90-1-1) and four other psorosis isolates from Argentina and the USA. In addition, CtRSV-4 particles were partially purified from local lesions in Chenopodium quinoa, and the preparations used to raise a rabbit antiserum. The antiserum was absorbed with extracts of healthy C. quinoa leaves, and a DAS-ELISA kit was prepared and tested for detection of CtRSV-4, CPsAV90–1-1, and other psorosis isolates from Argentina, the USA, Italy and Spain. The ELISA detected CtRSV-4 down to a tissue dilution of 1/1600, and most other psorosis isolates down to dilutions of 1/200–1/800. Three of a total of 20 heterologous isolates were consistently negative. Comparison of the PCR and ELISA results suggests that both methods can be used for detection of a range of psorosis isolates, but that variation of the viruses in the field might cause problems for any one diagnostic test.

Notes: Aggregation in the distribution of pathotypes of Erysiphe graminis f.sp. hordei, the barley powdery mildew pathogen, was investigated in field plots of ‘Golden Promise’, ‘Proctor’ and ‘Tyra’. ‘Golden Promise’ and ‘Proctor’ have no effective mildew resistance alleles, whereas ‘Tyra’ has Mla1, which was only effective against a proportion of the mildew population. Isolates of mildew were sampled according to a grid sampling scheme and their virulence spectra ascertained in order to group them according to pathotype. The populations were very diverse, and evidence for aggregation (quantified using join counts) was found only in the ‘Tyra’ plots, at distances of up to 1m. This aggregation was reduced in a subsequent sample. The results are consistent with a model in which mildew epidemics are started by a large number of initial infections, which then form diffuse, overlapping aggregations of clones. These aggregations then become more diffuse, so that the amount of aggregation reduces with time. The greater amount of aggregation seen in the ‘Tyra’ plots might have been caused by there being less initial inoculum with virulence towards that cultivar.

Notes: Zucchini yellow mosaic potyvirus (ZYMV), first isolated in Italy in 1973, described in 1981, and then identified in all continents within a decade, is one of the most economically important viruses of cucurbit crops. It is efficiently aphid-transmitted in a nonpersistent manner and it is also seed-borne in zucchini squash, which could have contributed to its rapid spread worldwide. Biological variability has been observed among ZYMV isolates, concerning host range, symptomatology and aphid transmissibility. More recent studies also revealed a serological and molecular variability. The survival of ZYMV in areas where cucurbits are not grown throughout the year remains to be elucidated, because very few natural over-wintering hosts have been identified so far. Partial control of ZYMV can be achieved by limiting transmission of the virus to the crops by aphids, using adapted cultural practices. Cross-protection with a mild strain has been shown to be effective against most ZYMV isolates. Resistance genes found in cucurbit germplasms are currently being introduced into cultivars with good agronomical characteristics. Pathogen-derived resistance strategies using the expression of ZYMV genes in transgenic plants have also been developed and appear promising. Nevertheless, the high biological variability of ZYMV justifies a careful evaluation of the deployment of genetic control strategies in order to increase their durability.

Notes: Survival and germinability of oospore populations of Peronospora viciae f.sp. pisi in soil were investigated. The percentage survival was assessed using the vital stain tetrazolium bromide. Germinability was defined as the percentage of oospores that germinated in water. Oospores 1–3 weeks old, embedded in plant tissue, were incorporated in a loamy sand or silt-loam soil and incubated at 3, 10 or 20°C or stored dry at 20°C and 30% RH. The percentage of surviving oospores in soil decreased rapidly with or after decomposition of the surrounding plant tissue at 10 and 20°C. After 29 weeks less than 10% of the oospores had survived. At 3°C, survival was 25% or more after 29 weeks. Germinability of the oospores was 3% at time of incorporation and had increased to a maximum of 50% after 4 weeks in the loamy sand soil. Increase in germinability of the dry-stored oospores was significantly later than that of the soil-incubated oospores. In soil, the initial increase in germinability was followed by a decline after decomposition of the surrounding plant tissue. In order to investigate survival of oospores under natural conditions, ground tissue or pod pieces of pea plants containing oospores 7 or 5 months old, respectively, were incorporated in two loamy sand soils or a silt loam soil in field plots. The surrounding plant tissue decomposed within 4 weeks and the percentage of surviving oospores decreased to less than 6% after a year. Survival of oospore populations was generally well described by the lognormal model, indicating that the risk of oospore death initially increased before decreasing later. Germinability of the oospore populations from ground tissue and pod pieces, 61 and 62% at incorporation, decreased rapidly after incorporation in soil. Oospores extracted from the silt-loam soil germinated poorly in water but caused high disease incidences in a bioassay, suggesting that oospores had become dependent on the host plant for germination.

Notes: Genetic diversity among Fusarium moniliforme isolates was analysed using vegetative compatibility group (VCG) and random amplified polymorphic DNA (RAPD) techniques. In the first experiment, RAPD was used to analyse a set of 43 isolates collected from different corn growing areas in Israel and the US. The isolates were assigned to 27 different VCGs. Thirty-two RAPD haplotypes were also detected by analysing 48 polymorphic bands. RAPD could differentiate all the VCGs, except in two cases where two VCGs were assigned a single RAPD haplotype. In six cases, however, molecular variation was detected among isolates belonging to the same VCG. Cluster analysis of the RAPD data showed a very good agreement with the VCG grouping, e.g. isolates of the same VCG were always closely clustered by the molecular data. In a second experiment, 63 isolates of Fusarium moniliforme were collected from six corn lines growing in a single corn field. Extensive genetic variation was observed among the isolates: 42 different VCGs and 37 RAPD haplotypes were identified. Once again, RAPD patterns could differentiate nearly all the VCGs. However, in four cases, two different VCGs were grouped into a single RAPD haplotype, while in another three cases, isolates of the same VCG could be differentiated by distinct molecular haplotypes. The variation data was used to gain insight on the population structure and the patterns of genetic variation among geographical locations and within a single field. Hierarchical gene diversity analysis of the RAPD data indicated that most of the genetic variability (81%) was distributed within corn lines in the same field, suggesting that RAPD haplotype, or VCG frequencies, were not significantly affected by the plant genotypes grown in this experiment. Most of the RAPD band combinations did not display significant gametic phase disequilibrium, suggesting that active recombination might be occurring in the field. Our results indicate that by using a small number of primers, similar resolution was obtained by RAPD and VCG analysis, respectively. RAPD analysis is however, simpler to perform and its sensitivity in genotyping individuals within Fusarium moniliforme can be increased by analysing more primers, enabling a more detailed population genetic analysis of this important pathogen.

Notes: A rapid, simple and reliable procedure was developed to evaluate biological control of Fusarium wilt of tomato by Penicillium oxalicum. The method consists in growing tomato plants in flasks with nutrient solution in a growth chamber. Plants were previously treated in the seedbed with a conidial suspension (107 conidia mL−1) of P. oxalicum 7 days before transplanting. Fusarium oxysporum f.sp. lycopersici (race 2) was added to the Hoagland solution just before transplanting. Different concentrations and several isolates of F. oxysporum f.sp. lycopersici were tested. Using this method, plants showed typical symptoms of the disease and the effect of the biocontrol agent was clear. Consumption of nutrient solution was reduced in diseased plants, and this reduction was diminished by treatment with P.oxalicum. Consumption of nutrient solution was correlated with other disease-related parameters (AUDPC, weight of aerial parts, stunting) and was an easy and objective parameter to measure.

Notes: Strains of Erwinia herbicola effective in the biocontrol of fire blight of hawthorn were used to investigate the possibility that the antagonistic activity is coded by plasmid-born genes. Agarose gel electrophoresis of isolated plasmids from four antagonistic Erw. herbicola strains showed a band of a supercoiled 12 kb plasmid in each strain, with a second band greater than 16.2 kb consistently seen in two strains. Erw. herbicola strains showed resistance to penicillin-G, which could be conferred on penicillin-G sensitive Escherichia coli TG1 by transformation with a pure Erw. herbicola plasmid preparation. Transformed strains of Esc. coli appeared to contain the Erw. herbicola 12 kb plasmid, but not the 〉 16.2 kb plasmid. In an agar plate assay, Esc. coli transformants produced an inhibition zone against Erw. amylovora similar to those produced by the original Erw. herbicola strains. In two biocontrol assays, the transformed Esc. coli strains had a suppressive effect on disease development on infected pear fruit slices and hawthorn blossoms.

Notes: Carica papaya, C. cauliflora and interspecific hybrids of these species were screened for resistance to two Australian isolates (338, 445) of papaya ringspot virus-type P (PRSV-P). Plants were manually inoculated with PRSV-P in the glasshouse and the reaction assessed 30 days later by back-inoculation to susceptible Cucurbita pepo and by a plate-trapped antigen-enzyme linked immunosorbent assay (PTA-ELISA). Both parents and interspecific hybrids were also planted adjacent to infected C. papaya and 30 days later tested for PRSV-P by PTA-ELISA. All interspecific hybrid and C. cauliflora plants manually inoculated in the glasshouse or planted in the field failed to become infected, whereas C. papaya plants, in both situations, were infected by PRSV-P. In addition, the surviving interspecific hybrid and C. cauliflora plants tested negative, while all C. papaya plants were positive for PRSV-P in both the back-inoculation and PTA-ELISA tests. Thus, the interspecific hybrid and C. cauliflora plants were resistant to the Australian PRSV-P isolates.

Notes: Twelve diverse cacao (Theobroma cacao) genotypes were assessed for pod resistance to Phytophthora palmivora at the penetration and post-penetration stages of infection using two inoculation methods. Correlation analysis between a number of pod characteristics (stomatal frequency, stomatal pore length, surface wax, thickness of exocarp/endocarp, hardness of exocarp/mesocarp, moisture content) and resistance indicated a strong relationship between resistance to lesion establishment (lesion frequency) and the joint effect of stomatal frequency and pore length. The epidermal impressions of the pod surfaces bearing germinating zoospores of P. palmivora provided evidence that penetration occurs through stomata, epidermal hair base, scar and by direct penetration. A poor correlation was obtained between the pod characteristics studied and post-penetration resistance, suggesting that this resistance, assessed by lesion size, is not governed by morphological or physical characteristics of the pod, but probably by biochemical factors. The importance of these findings in breeding of cacao for resistance to P. palmivora is discussed.

Notes: A nonpathogenic Erwinia amylovora transposon mutant that has an insertion in the guaB gene was isolated. The mutation results in a nutritional requirement for guanine or xanthine, and loss of ability to produce ooze on immature pear fruit and to cause symptoms in the apple seedling assay. The mutant expressed other known virulence determinants including extracellular polysaccharide and had an intact hrp/dsp cluster. In addition it was able to grow in host tissue, although the population size in planta was maintained at a considerably lower level than that seen with the parent strain. The inability of the Erwinia amylovora guaB mutant to cause disease indicates that levels of guanine in plant tissue are likely to be insufficient to maintain optimal growth via the purine salvage pathway. This, in turn, appears to compromise the ability of the mutant to develop a sufficiently large population size in planta to overcome host defence mechanisms and cause disease symptoms. This indicates that a functional de novo guanine synthetic pathway is important for Erwinia amylovora to grow on plant tissue and cause disease.

Notes: Vegetative compatibility of strains of Diaporthe ambigua has not previously been examined. Single ascospore and single ascus strains, originating from individual apple, pear and plum rootstocks, were paired on freshly prepared oatmeal agar to determine if vegetative incompatibility could be detected in D. ambigua. Barrage reactions were evident as black lines along the zone of mycelial contact between expanding colonies (vegetative incompatibility reaction). Strains from cankers within an area were of numerous vegetative compatibility groups (VCGs). Strains from adjacent rootstocks usually differed in VCG. D. ambigua has the ability to outcross, and does so, despite its apparent homothallic nature. DsRNA-containing strains of D. ambigua developed a broad, clear zone when paired with a dsRNA-containing strain from a different VCG.

Notes: One major gene for resistance to isolate WYR 85-22 of race 6E0 of Puccinia striiformis was identified by genetic analysis of the differential cultivars Heines Peko, Strubes Dickkopf and Heines VII. This gene was different from Yr2Yr6 already identified in Heines Peko (Yr2Yr6) and Heines VII (Yr2), was allelic in the three cultivars and also to a gene expressed in the other differentials Reichersberg 42 (Yr7) and Clement (Yr9). In Heines Peko, Strubes Dickkopf and Heines VII, a minor gene was also postulated, which, it is proposed, gave only a low level of resistance by itself but strengthened the expression of the major gene when the latter was homozygous or heterozygous. The genetics of the resistance was analysed using six resistance classes and applying multidimensional analyses. The number of resistance genes was hypothesized using data from F3 families from crosses between the three differentials and the cultivar Heines Kolben, which is susceptible to this race.

Notes: Phytophthora root rot is of paramount importance in avocado orchards of southern Spain. Soil solarization has been demonstrated to control the pathogen in infested areas from which infected trees had been removed. We aimed to determine whether soil solarization in established avocado orchards controls the disease. Soil solarization increased average maximum hourly soil temperatures by 6.5–6.9°C in unshaded areas of avocado orchards in coastal areas of southern Spain, depending on depth and year. The corresponding temperatures in shaded areas were c. 2–3°C lower. P. cinnamomi in soil, on infected avocado rootlets, and in a nutrient substrate buried at 30–60 cm depth was reduced to negligible amounts after 6–8 weeks of solarization in both unshaded and shaded locations of avocado orchards. P. cinnamomi could not be detected in avocado rootlets up to 14 months later, suggesting a long-term effect. Soil solarization did not affect growth of the trees, and fruit yields were increased as compared with control plots. Following soil solarization for 3 weeks from mid-July 1994, when maximum hourly temperatures reached 33–36°C, P. cinnamomi could not be recovered from a depth of up to 45 cm in unshaded areas or from a depth of up to 30 cm in shaded areas after the initial 10-day period. The viability of inoculum of the pathogen buried at depths between 15 and 60 cm in bare soil was determined by sequential sampling in two solarization experiments starting 12 June and 4 July 1995, respectively. In the first experiment, P. cinnamomi could not be detected at any depth after 4–8 weeks of solarization in unshaded areas but could be recovered at all depths except 15 cm in shaded areas. In the second experiment, where temperatures were higher and the soil surface not shaded, P. cinnamomi could not be recovered after 2 weeks at 15 and 30 cm.

Notes: Genetic data showed that a common gene was present in the three differential wheat cultivars Heines VII (Yr2), Heines Peko (Yr2, Yr6) and Heines Kolben (Yr6) expressed against the race 109E9 of Puccinia striiformis which possesses virulence for Yr6 and avirulence for Yr2. This supported the hypothesis that Heines Kolben carries the gene Yr2 in addition to Yr6 for resistance to yellow rust in a genetic background in which Yr2 is weakly expressed in seedlings. F1, F2 and F3 progenies from the cross Heines Kolben × Peragis confirmed the monogenic segregation of Yr2 in Heines Kolben and demonstrated the variation of its expression with environmental conditions.

Notes: Erysiphe sp. is a causal agent of powdery mildew on Rhododendron. A novel in vivo method permitting the screening of fungicides on woody plants is described. Eight fungicides were evaluated for activity against Erysiphe sp. using Rhododendron ponticum microplantlets grown in vitro. Pathogen development changed with both the type of fungicidal compound and the concentrations applied. The most active materials were fenpropidin and penconazole, which showed high activity at the lowest concentrations. Six of the compounds performed more effectively than a mixture of bupirimate + triforine (Nimrod T), the standard recommendation for control of this pathogen on Rhododendron. All fungicides affected the sporulation of Erysiphe sp., with propiconazole, pyrazophos and triadimenol causing a significant increase in sporulation at the lowest concentrations. At higher concentrations, sporulation was significantly reduced by all treatments. No phytotoxic effects were detected with any fungicide at any concentration. The growth of plantlets in most treatments showed no significant difference from the untreated controls. The results of the study are discussed in relation to strategies for control and the epidemiology of Erysiphe sp. infecting Rhododendron.

Notes: Solarization trials were carried out over 3 years and in two countries to control crown gall disease on fruit trees and eliminate Agrobacterium. In 1992, agrobacteria in naturally infested soils of two Italian nurseries were monitored before and after solarization. Agrobacteria populations decreased by 99% and 92% after the treatment; however, crown gall incidence did not decrease. In 1993 and 1994 solarization was tested in Oregon in fields artificially infested with two marked strains of A. tumefaciens. In sandy loam soil, the target bacteria were eliminated in 4 weeks, while in silty clay soil the populations were markedly reduced after 2 months of treatment. Crown gall incidence on cherry rootstocks transplanted to the field at the end of 1993 was 3.7% in the sandy loam soil control plots, while no tumours were observed on plants from solarized plots. The use of solarization in combination with reduced doses of metham-sodium was also evaluated.

Notes: Glasshouse experiments to test the activity of commercial fungicides against Septoria tritici were carried out under controlled conditions. In addition to the parameter, % necrotic leaf area (NEC), used to estimate the pathogen-induced leaf damage, the number of pycnidia per leaf (PYC) was determined to quantify the pathogen itself. Curative fungicide treatments were applied 100–210 day degrees after inoculation. A high curative activity was achieved up to 170 day degrees after inoculation, whereas the treatments at 200–205 day degrees were less effective. The best curative activity was observed for epoxiconazole-based treatments, followed by slightly less active azoles in the ranking tebuconazole, cyproconazole, prochloraz and flusilazole. Greater differences between the fungicides were observed for the protectant fungicide properties, which were tested 50–350 day degrees prior to inoculation. The best persistency was observed for epoxiconazole, whereas tebuconazole, cyprocoazole, prochloraz and flusilazole showed declining activity with this ranking. Combinations of triazoles with the active ingredients kresoxim-methyl and chlorothalonil, which are known to inhibit spore germination, significantly improved the longevity of the remaining green leaf area in comparison with disease-free treatments with triazoles alone. The results obtained under glasshous conditions were compared with field studies on S. tritici development after treatments with tebuconazole to place the results in context. The comparison of the assessment parameter PYC and NEC between glasshouse and field trial showed that curative and protectant fungicide properties based on microscopic assessments of PYC in the glasshouse correlated well with results from field trials.

Notes: The genotypic diversity in a South African population of Fusarium subglutinans f.sp. pini (F.s. pini) was determined, based on the number of vegetative compatibility groups (VCGs). Isolates of F.s. pini from South Africa (69), California (five) and Florida (19) were included in the study. The nit1 (or nit3) and NitM mutants were selected as chlorate resistant sectors and paired on minimal medium. The South African isolates of F.s. pini were assigned to 23 different VCGs. No heterokaryons formed between isolates from South Africa, California and Florida. The high degree of genotypic diversity in the South African population of F.s. pini is probably due to some level of sexual reproduction in the population.

Notes: Yellows-diseased plants of Crepissetosa (hawksbeard), Knautiaarvensis (field scabious), Convolvulusarvensis (field bindweed), Picrisechioides (bristly oxtongue), Echiumvulgare (blueweed) and Calendulaofficinalis (pot marigold) collected in central and southern Italy were examined for phytoplasma infection by means of polymerase chain reaction (PCR) technology using universal phytoplasma primers directed to ribosomal sequences. The detected phytoplasmas were characterized and differentiated using restriction fragment length polymorphism analysis of PCR-amplified DNA. The phytoplasma detected in diseased pot marigold plants was identified as a member of the aster yellows group and proved indistinguishable from a strain of the American aster yellows phytoplasma. The phytoplasma identified in diseased field bindweed plants is a putative new type of the stolbur group that differed from the typical stolbur phytoplasma. Phytoplasmas detected in diseased hawksbeard, blueweed and field scabious plants are all putative new members of the sugarcane white leaf group while the phytoplasma detected in diseased bristly oxtongue plants represents a new member of the faba bean phyllody group. For hawksbeard and field scabious this is the first report on the occurrence of phytoplasma diseases, whereas phytoplasmas infecting bristly oxtongue and blueweed have never been characterized before.

Notes: An isolate of Gliocladium roseum proved highly antagonistic to Botrytis cinerea. Sporulation of B. cinerea on chickpea seed naturally infected or inoculated with B. cinerea was suppressed by seed treatment with conidial suspensions of G. roseum at 107 and 108 conidia/mL, respectively. Establishment of healthy seedlings in punnets (small trays) 5 weeks after sowing with inoculated seed was increased from 29.2% to 59.7% by treatment with G. roseum at 3×107 conidia/mL, and from 1.4% to 69.4% with G. roseum at 3×108 conidia/mL, the latter being equivalent to disease control by Thiram. There was no significant effect of Rhizobium on disease suppression by G. roseum, and treatment with G. roseum at 108 conidia/mL did not reduce nodulation. Amendment with culture filtrates of G. roseum did not affect the growth rate of B. cinerea on potato dextrose agar, indicating that constitutive production of an antibiotic is not involved in biocontrol. A selective medium was developed to enumerate propagules of G. roseum on seed recovered from soil. There was no significant change in the population of G. roseum on seed after incubation for 4 weeks in soil to which the isolate of G. roseum was indigenous.

Notes: The pathogenic variability of the barley scald fungus, Rhynchosporium secalis, in central Norway was examined in 1994. The climate in this region is usually cold and wet during the growing season of spring barley. Leaf blotch is prevalent and causes significant yield losses. Forty-two isolates of the fungus, from naturally infected spring barley in four counties, were differentiated into 32 pathotypes by the standard differential set for R. secalis. All pathotypes were complex and had virulence for nine to 22 differentials. The cultivar Osiris was resistant to all isolates tested. The cultivars C.I.8162, Hudson, Atlas 46 and C.I.3515 were resistant to the majority of the isolates. Several differentials with various resistance genes were susceptible to up to 100% of the isolates. Isolates were derived from local cultivars with no known resistance genes, suggesting that R. secalis populations in central Norway are characterized by a high degree of seemingly unnecessary pathogenicity. Because of the great variability and complexity of the pathotypes, traditional breeding methods using single major genes are not likely to be effective in central Norway.

Notes: The effect of soil moisture content on the suppression of Rhizoctonia stem canker on potato by mycophagous soil animals was studied in growth chambers. Three soil moisture levels were established in two bioassays, in which potato sprouts grew through a 15-cm soil layer inoculated with sclerotia of Rhizoctonia solani (AG-3). In one experiment two levels of R. solani inoculum were applied. The effect on plant disease of mycophagous soil fauna was assessed by adding the springtail Folsomia fimetaria and/or the nematode Aphelenchus avenae to the soil. In the absence of mycophagous organisms, Rhizoctonia disease severity on potato stems was highest in dry soil. A. avenae and F. fimetaria reduced Rhizoctonia stem canker when applied at populations found in the field. They were effective over a broad range of soil moistures. The stimulatory effect of dry soil conditions on Rhizoctonia stem canker was counteracted by a greater efficacy of the mycophagous soil fauna under these conditions. Mild drought stress did not seem to be a limiting factor in the biological control of stem canker by these two organisms.

Notes: Stem culture in vitro was attempted for the elimination of mulberry dwarf phytoplasma. Stem segments, from mulberry plants (Morus alba) seriously infected with the phytoplasma, were cultured on Murashige and Skoog (MS) solid nutrient medium without phytohormones for 2–3 months. The tissue cultures, and regenerated plants grown under greenhouse conditions for 3 years, were tested by polymerase chain reaction (PCR) and 4,6-diamidino-2-phenylindole (DAPI) staining for the presence of the phytoplasma, and also observed for mulberry dwarf symptoms. From 70% to 90% of regenerated plants were apparently freed from the phytoplasma since they remained PCR-negative, DAPI-negative and symptom-free for as long as tested (3 years).

Notes: On oilseed rape, 207 leaf lesions attributed to Leptosphaeria maculans were classified as typical or atypical. Starch gel electrophoresis of glucose phosphate isomerase (GPI) performed on extracts of 229 leaf lesions comprising the 207 with L. maculans symptoms and 22 with Pseudocercosporella capsellae symptoms, yielded four different electrophoretic patterns of alloenzymes designated ET1 to ET4. In addition to ET1 and ET2, characteristic respectively of A- (highly virulent) and B- (weakly virulent) group isolates of L. maculans, the previously undescribed ET3 allozyme was recovered from a few typical and atypical L. maculans leaf lesions. The fastest ET4 allozyme was specific to P. capsellae. All but two typical leaf lesions produced the ET1 allozyme, whereas atypical lesions produced one of the three L. maculans allozymes. Occasionally a mixture of two allozymes was recovered from a same-leaf lesion. GPI electrophoresis performed directly on leaf lesions proved a useful and reliable method to identify L. maculans, and to differentiate between L. maculans and P. capsellae. This method of discrimination enabled deductions, from 377 leaf lesions analysed, about the structure of L. maculans populations on different oilseed rape varieties.

Notes: Mycocentrospora acerina, the causal agent of liquorice rot of carrot roots, produces several cell-wall polysaccharide degrading enzymes in vitro. To assess the involvement of these enzymes in tissue invasion, the production and localization of pectin methylesterases, polygalacturonases, pectate lyases and endoglucanases were measured in root tissue infected by M. acerina. Isoelectrofocusing studies demonstrated the production of three isoforms of pectin methylesterase in healthy tissue. In infected tissue, two isoforms already observed in the culture filtrate and two novel isoforms were detected. Tissue maceration was associated with in vivo production of pectin methylesterase and polygalacturonase, which were first detected on the second day whereas pectate lyase and endoglucanase activities were detected only from the fourth day after infection. Maceration was always detected ahead of the mycelium, indicating diffusion of these enzymes.

Notes: Pseudomonas fluorescens strain CHA0 suppresses various plant diseases caused by soil-borne fungi. The pseudomonad produces the antimicrobial metabolites 2,4-diacetylphloroglucinol (Phl), pyoluteorin (Plt) and hydrogen cyanide, which are important for disease suppression, as well as the siderophores pyoverdine (Pvd), salicylic acid (Sal) and pyochelin (Pch). In the current work, a derivative of CHA0 with a mutation in the global regulator gene gacA (GacA−), which is unable to produce Phl, Plt and HCN, failed to protect the dicotyledonous plants cress and cucumber against damping-off caused by Pythium ultimum. In contrast, the GacA− mutant could still protect the Gramineae wheat and maize against damping-off mediated by the same strain of P. ultimum, and wheat against take-all caused by Gaeumannomyces graminis. However, the GacA− mutant overproduced Pch and Pvd. To gain more insight into disease protection afforded by the GacA− mutant, a GacA− Pvd− double mutant (strain CHA496) was constructed by gene replacement. Strain CHA496 overproduced Pch and Sal compared with CHA0 and protected wheat against P. ultimum and G. graminis, whereas cress and cucumber were not protected. Addition of FeCl3 repressed Pch and Sal production by strain CHA496 in vitro and impaired the protection of wheat in soil microcosms. In conclusion, a functional gacA gene was necessary for the protection of dicotyledons against root diseases, but not for that of Gramineae. Results indicated also that Pch and/or Sal were involved in the ability of the GacA− Pvd− mutant of CHA0 to suppress root diseases in Gramineae.

Notes: The present study shows that a large range of potato cultivars (29/33 tested), widely grown in the world, are susceptible to potato tuber necrotic ringspot disease caused by potato virus Y. The three factors studied in this work, which proved to influence the level of tuber necrosis reaction, were, first, the plant genotype, since varietal behaviour exhibited large differences; second, the virus genotype, since variations of virulence occurred between the four isolates tested; and third, the environmental conditions, as shown by the different rates of tuber necrosis obtained under contrasting conditions of temperature as much during the growing period as during storage. Three of the cultivars tested, Spunta, Maris Piper and Thalassa, failed to produce necrotic tubers, although infected with a virulent tuber-necrosing isolate. This result, following observations on the inheritance of the tuber necrosis trait, suggests that at least a major dominant gene controls this reaction in non-sensitive cultivars. On the other hand, the extreme resistance genes (Ry) provide a good resistance to virus infection, thus, preventing tuber necrosis under field conditions.

Notes: Six wheat genotypes with wide genetic variability for resistance to bacterial leaf streak were crossed in diallel fashion to determine the inheritance of resistance to this disease. Parental genotypes and their F1 hybrids were inoculated at the second leaf stage with an Iranian isolate of Xanthomonas campestris pv. cerealis. Two experiments were undertaken in a controlled environment chamber. Results showed that the genotypes IBPT66, IBPT84 and IBPT34 had a high level of partial resistance to the disease. General and specific combining abilities presented several significant positive or negative values, showing the importance of both additive and dominant genetic control for partial resistance to this bacterial disease. The resistant line IBPT66 could be a good donor of partial resistance to bacterial leaf streak in wheat.

Notes: Formulations of bacterial biocontrol agents were evaluated for the control of pea root-rot caused primarily by Pythium ultimum and Rhizoctonia solani, at different levels of disease severity in field trials. Pseudomonas fluorescens (strain PRA25) in a peat-based formulation increased yield by 17% over the untreated, in a trial with light disease infection, and by 120% in another with moderate infection. Other bacteria including P. cepacia (strain AMMD) and fluorescent pseudomonads increased seedling emergence, and decreased disease incidence and severity, but with variable effects on yield when disease level was light to moderate. Biocontrol agents resulted in only limited control when disease was severe. Control with Captan did not differ significantly from that obtained with bacterial biocontrol agents. The application of Rhizobium granular inoculum together with PRA25 granules did not differ from other treatments in disease control, indicating that Rhizobium is compatible with biocontrol agents. The population dynamics of a fluorescent pseudomonad introduced into the rhizosphere in peat or granular formulation was monitored using an antibiotic-resistant mutant marker strain. The bacterium in peat formulation established a considerably higher population than that in granular formulation in 1993 trials, and a slightly higher population in 1994 trials. The higher population may have been responsible for the efficiency of root colonization and the effectiveness of disease control.

Notes: In controlled environment experiments, when oilseed rape pods or leaves were inoculated with spore suspensions of Alternaria brassicae, the maximum disease incidence (proportion of pods or leaves diseased) increased as wetness period after inoculation increased from 4 to 24 h and as temperature increased to 20°C. There was a clear relationship between disease incidence on pods and incidence on leaves with the same wetness/temperature conditions. Logistic equations described the effects of wetness period after inoculation on disease incidence (number of pods or leaves infected) or disease severity (number of lesions on pods or leaves) using temperature-dependent and tissue-dependent parameters. The time from inoculation to the appearance of the first lesions was shorter on pods than on leaves at temperatures ≤15°C and wetness periods ≤12 h. Two-dimensional response surface equations or simple interpolations from one-dimensional equations were used to develop contour maps of expected disease incidence and severity, respectively, on leaves or pods to estimate the effects of different combinations of wetness period during infection and temperature on disease development.

Notes: Genetic variation of the barley powdery mildew fungus (Erysiphe graminis f.sp. hordei) was estimated in three Danish local populations. Genetic variation was estimated from the variation amongst clones of Egh, and was therefore an estimate of the maximum genetic variation in the local populations. The average gene diversity, Ĥ, was estimated as 0.84. The effective population size was estimated as: log10 (N^e) = 0.64 − log10(μ), or 4.4 × 109, assuming a nucleotide mutation rate ( μ) of 10−9 per base per generation. There was no significant genetic differentiation between locations.

Notes: The effects of 21 weeds acting as hosts of tomato spotted wilt virus (TSWV), temperature, thrips population and diversity on disease progress in chrysanthemum cv. Polaris were studied. Under greenhouse conditions, only Taraxacum officinale, Bidens sp., Resedaluteola and Mirabilis jalapa were hosts for TSWV. Of 38 weeds species in the area surrounding a chrysanthemum field, Tithonia tubaeformis and R. luteola had the highest populations of adult and immature thrips. These weeds, as well as M. jalapa, had an extensive seasonal distribution and may play a key role in the disease progress. Seventeen thrips species belonging to the genera Bravothrips, Thrips and Frankliniella were identified on weed flowers, with Frankliniella occidentalis (FOC) representing 9.5% of all thrips identified. Of 123 thrips collected from chrysanthemum inflorescences, 9.75% were FOC, and only 2.5% of them transmitted TSWV. Of all the thrips species collected from chrysanthemum flowers in the field, only FOC was capable of transmitting TSWV. On 120 experimental plots established at two sites, with three transplanting dates (June, July and August), it was estimated that 1.25% of the chrysanthemum cuttings were already infected with TSWV when transplanted. Secondary spread, vectored by FOC, occurred only for the earliest transplanting date and resulted in a further 2.36% disease incidence.

Notes: The occurrence of grapevine leafroll-associated virus 1 (GLRaV-1), grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine virus A (GVA) was demonstrated in a viticultural region of northern Italy (Emilia-Romagna) using immunoelectron microscopy. Virus incidence was subsequently assessed using ELISA. A total of 60.6% of the 150 clone selections tested, from 18 local Vitis vinifera cultivars, were found to be infected. ELISA did not reveal the presence of grapevine leafroll-associated virus 2 (GLRaV-2) or grapevine leafroll-associated virus 5 (GLRaV-5). GLRaV-1, GLRaV-3 and GVA were found individually and in various combinations. The most common findings were GLRaV-1 alone (25.3%) and associated with GVA (33%). Serological data confirmed that the majority (91%) of the clones known to be affected by grapevine leafroll (GLR), on its own or in association with rugose wood (RW), contained viruses. On the other hand, where the RW phenomenon was present on its own, only 40% of these clones were ELISA-positive. The implications for the biology of GLR and RW are discussed and the complex aetiology of these grapevine diseases is confirmed.

Notes: Inadequate species definitions present a serious problem to the pathologist working in plant hygiene or quarantine which demands urgent attention. Species concepts in the downy mildews have not kept pace with developments in evolutionary and molecular biology, or with advances in ecological genetics, because the downy mildews are obligate biotrophs that are not easily cultured in the laboratory. Existing approaches to species concepts in this group (morphometric, Ga¨umann’s ‘biological species’ and Skalicý’s ‘eco–physio–phentic’ concepts) are examined and found to be inadequate and potentially misleading. The systematic treatment of the downy mildews is beginning to benefit from the application of modern methods of systematic analysis. The contribution and potential of ultrastructure, karyotyping, sterol and fatty acid composition, isoenzyme patterns, molecular biology, numerical methods, immunoassay and hypotheses of coevolution to the development of species concepts are reviewed and their wider application is seen as a priority. The application to the downy mildews of two widespread species concepts, the biological and phylogenetic concepts, is examined in the light of the information gained from modern methods of analysis, but neither is found adequate to describe species of downy mildews as they occur in nature. Modern methods can suggest phylogenetic relationships on the basis of statistical probabilities and may also detect microevolutionary change, but it is concluded that much more information is required about individual breeding systems, gene flow, ecology, phylogeny and distribution before informed decisions about the delimitation of most species can be made. Until patterns of genetic diversity can be established, a modified phylogenetic species concept may offer one interim solution to the problem of species definition in the downy mildews.

Notes: About 90 barley cultivars mostly of European or Japanese origin, were grown for 2–5 years at eight sites in China where barley yellow mosaic virus was known to occur. The sites were selected because they had previously been used to screen breeding lines and some differences between them in cultivar response had been suspected. ELISA tests showed that symptomless plants were not infected by the virus and the proportions of plants with symptoms were therefore recorded as a measure of susceptibility. European cultivars carrying the ym4 gene, which confers resistance to the common European strain, were usually resistant at two sites but susceptible at the others, but one (cv. Energy) was resistant at all sites. Eleven of the Japanese cultivars showed differential responses between sites but there was no correspondence with strains recognized in Japan. There are probably several distinct Chinese strains but further experiments would be needed to identify them. The Japanese cultivars Chosen, Hagane Mugi, Iwate Mensury 2 and Mokusekko 3 seem to be resistant to all known virus strains and are probably the most useful for plant breeders.

Notes: The infection process of a Colletotrichum species causing latent infection and anthracnose in cowpea (Vigna unguiculata) was studied in seedlings by light and confocal microscopy. Leaf surfaces were extensively colonized by an anastomosing network of germ-tubes and conidia. This epiphytic mycelium produced abundant secondary conidia on short conidiophores. Although melanized appressoria were developed, the host surface was not penetrated directly. The fungus only gained ingress into leaves through stomatal openings, by means of undifferentiated germ-tubes, and slowly colonized the mesophyll by intercellular hyphae, without initially producing visible symptoms. Anthracnose lesions with multisetate acervuli appeared on senescent leaves after a prolonged symptomless period of host colonization lasting 〉 2 weeks. Analysis of the nucleotide sequences of the amplified D2 and ITS-2 regions of rDNA revealed close similarities (95–96%) between this cowpea pathogen and isolates of C. gloeosporioides from Aeschynomene virginica,Stylosanthes scabra and Mangifera indica. These results, in addition to other morphological and growth attributes, identify this endophytic anthracnose pathogen of cowpea as a Colletotrichum species distinct from C. capsici and C. destructivum.

Notes: Fungi, with spores characteristic of the genus Alternaria, were isolated from necrotic lesions on leaves of cabbage, cauliflower, Chinese kale and choi-sum growing in Thailand, and were proved by Koch's postulates to be the causal agents of a disease known as dark leaf spot. All isolates corresponded in morphology to descriptions of Alternaria brassicicola and the identification was confirmed by analysis of the ITS1, 5.8S gene and ITS2 regions of rDNA, the nucleotide sequences of isolates from all four plants being identical to each other and to the published sequence of a known isolate of A. brassicicola. Culture filtrates of isolates of the fungus from each host, grown on a defined medium consisting of Czapek–Dox nutrients supplemented with cations, were toxic to cells isolated from the four host plants. Taking the data overall, filtrates from the cauliflower isolate were significantly less toxic than those from the other isolates. Although the filtrate from the cabbage isolate was most toxic to cabbage cells and that of the choi-sum isolate most toxic to choi-sum cells, filtrates of the Chinese kale and cauliflower isolates were most toxic to cells of plants other than those from which they were isolated.

Notes: Stem base disease (eyespot, sharp eyespot and brown foot rot) was assessed visually and by the polymerase chain reaction (PCR) technique on single plants sampled at four-week intervals in two crops of winter wheat grown in the UK in 1992–3. PCR assays were conducted for Fusarium avenaceum, F. culmorum, both varieties of Microdochium nivale, both eyespot-causing species of Tapesia and Rhizoctonia cerealis. PCR diagnoses were compared with visual diagnoses at each time point. Eyespot was caused principally by T. acuformis (R-type) and developed rapidly late in the season. Visual diagnoses of eyespot were largely confirmed by PCR but T. acuformis was detected in many plants lacking eyespot symptoms. R. cerealis was detected at relatively low incidences on both sites, and sharp eyespot visual diagnoses did not correlate with the incidence of any of the pathogens assayed by PCR. Brown foot rot, caused principally by Microdochium nivale var. majus, accumulated earlier in the season than eyespot. Overall, visual diagnoses of stem base disease coincided poorly with PCR data until after growth stage (GS) 30.

Notes: The inheritance of partial resistance to race 2 of Albugo candida was studied in a canola-quality line of Brassica juncea. This partially resistant line was crossed with the susceptible B. juncea cultivar Commercial Brown. F1, F1(reciprocal), F2, BC and doubled haploid generations from the cross were inoculated with a zoospore suspension of race 2 to study segregation of partial resistance. The partially resistant phenotype appeared to be controlled by a single dominant gene that has variable expression. This partial resistance can have implications in breeding for disease resistance against white rust, as adult plants did not develop hypertrophic growth or stagheads under greenhouse and field conditions.

Notes: Citrus psorosis is a serious and widespread disease associated with citrus psorosis virus (CPsV), a novel filamentous negative-stranded virus in the genus Ophiovirus. Laborious and costly indexing on test plants has been the only routine diagnostic method available, but recently an antiserum usable in double antibody sandwich (DAS) ELISA has been prepared. Here, major improvements to the DAS-ELISA protocol, a new purification method, and production of two monoclonal antibodies (mabs) to CPsV, an IgG and an IgM are reported. A highly sensitive triple antibody sandwich (TAS) ELISA making use of the mabs is described. In glasshouse citrus the homologous virus was still detectable at a tissue dilution of 1/6250 in DAS and at 1/31250 in TAS-ELISA. Both the DAS and IgG mab-TAS formats detected all CPsV isolates so far tested (from Argentina, Italy, Lebanon, Spain and the USA). A few isolates were not detected by the IgM mab.

Notes: Three methods were compared to assess the susceptibility of vegetatively propagated chrysanthemum to tomato spotted wilt tospovirus (TSWV): mechanical and thrips-mediated inoculation of whole plants, and a leaf-disc assay. As symptom expression was often poor or even absent, TSWV infections and subsequent susceptibility to TSWV were determined by ELISA. All 15 chrysanthemum cultivars tested were susceptible to TSWV, irrespective of their degree of vector resistance (based on feeding-scar damage rates). Thrips-mediated inoculation using different numbers of thrips revealed that 100% infection was obtained when plants were challenged by six thrips per plant, whereas 80 and over 50%, respectively, of the plants became infected when inoculated by a single male or female thrips. However, false negatives were scored even after intensive sampling because of erratic, cultivar-specific and time-dependent virus distribution after inoculation in the plants. Labour-intensive samplings and long incubation periods could be overcome by a readily applicable leaf-disc assay. This assay was as reliable as thrips-mediated inoculation of whole plants, and its use is therefore favoured to assess chrysanthemum cultivars for TSWV susceptibility.

Notes: The transmission of tomato spotted wilt tospovirus (TSWV) by Thrips tabaci collected from leek was studied using the petunia local-lesion leaf-disc assay. After an acquisition-access period of 72 h given to newborn larvae up to 8 h old, the efficiency of transmission by adults was determined in three inoculation-access periods of 48 h. This efficiency varied for six T. tabaci populations from 0.7 to 11.6% in experiments using the Greek TSWV isolate GR-04. Males were more efficient transmitters than females (19 out of 176 versus five out of 494). Frankliniella occidentalis transmitted the same virus with a higher efficiency (34.8%). The transmission rate differed also among TSWV isolates, as shown in tests with four T. tabaci using two isolates. The virus was more efficiently acquired from infected leaf material of Datura stramonium than from that of Emilia sonchifolia. Plants of the latter species were more susceptible than Nicotiana tabacum in thrips transmission tests.

Notes: Experiments were conducted over two seasons (1995 and 1996) to determine the survival of Botrytis cinerea conidia applied to the fruit surface of kiwifruit (Actinidia deliciosa, cv. Hayward). In 1995, potted vines were exposed for nine weeks to controlled environments at the National Climate Laboratory and to natural conditions in a shadehouse. In 1996, field experiments were carried out over a period of 16 weeks at two research sites. Change in number of conidia (enumeration), viability (germination) and vigour (germ-tube length, 1995; infection of host tissue, 1996) of conidia were assessed over time. In both years, the number of conidia/fruit declined significantly over time. However, conidia remained viable on the fruit surface throughout the two experiments. In both seasons, approximately 40% of the conidia recovered still germinated after field exposure and the vigour of viable conidia remained constant over the duration of the two experiments. The results of the research showed that B. cinerea conidia are able to survive on fruit surfaces of kiwifruit, remaining viable and infectious throughout the growing season.

Notes: Cold tolerant isolates of Gaeumannomyces graminis var. graminis (Ggg) and Phialophora sp. (lobed hyphopodia), which produced at least comparable growth rates at 5°C to those of pathogenic G. graminis var. tritici (Ggt), were shown to control take-all disease in wheat effectively in 2 years of field experiments in New South Wales, Australia. The addition of oat inoculum of these fungi at the rate of 60 kg/ha to the seeding furrow significantly (P ≤ 0.05) reduced disease and increased grain yields by 33–45% compared to the Ggt alone treatment. The use of 30 kg/ha of oat inoculum also significantly (P ≤ 0.05) reduced disease and increased grain yields by 21–44%. These high levels of take-all control were obtained consistently from four field experiments on three different soil types with different pHs. A treatment inoculated with Ggg alone showed no disease symptoms and produced grain yields similar to that of untreated wheat. This fungus is, therefore, non-pathogenic to wheat. At high rates of inoculation of Ggg and Phialophora sp. (lobed hyphopodia), 65–80% of tillering wheat plants (GS 32) had root systems colonized by these fungi. In contrast, two Pseudomonas spp. and an isolate each of Ggg and Phialophora sp. (lobed hyphopodia), which did not grow at 5°C, were ineffective in controlling take-all. Take-all assessments during heading (GS 61-83) were highly correlated (R2=0.6047, P≤0.0005) with the relative yield increase or decrease of inoculated treatments compared to the Ggt alone treatment. The use of a Ggg isolate (90/3B) and a Phialophora sp. (lobed hyphopodia) isolate (KY) for take-all control has been patented. These fungi are being developed for commercial use.

Notes: Squash blotting on nitrocellulose membranes was used to detect tomato spotted wilt virus (TSWV) in individual Frankliniella occidentalis adult thrips using a specific polyclonal antiserum. This method offers a simple and reliable procedure to test a large number of thrips and was less time-consuming than alternative techniques available. A 95% agreement was found between the results of squash-blot and plant transmission assay. Based on this agreement, this technique was used to study the relative number of viruliferous thrips in F. occidentalis field populations collected from a farm in Catalonia where TSWV causes important economic losses. The percentages of viruliferous individuals detected from a total of 1509 collected thrips increased from 0% to 2% in the different surveys conducted in the 1993 growing season. Squash-blot proved to be a rapid and inexpensive technique to screen TSWV viruliferous thrips from field populations. The results obtained by this method could be used together with other epidemiological data to forecast TSWV-induced diseases.

Notes: Inoculation of celery plants with viruses CV036 (celery mosaic) and CV506 (parsnip yellow fleck) decreased blight on leaves inoculated later with Septoria apiicola by 17–39% and 54–91%, respectively. There was a significant negative correlation between the amount of blight and the furanocoumarin content of the celery petioles. The antifungal activity of these furanocoumarins was demonstrated by their in vitro inhibition of the germination of spores of S. apiicola and Botrytis cinerea. Scanning electron microscopy of virus-infected leaves sprayed with spores of S. apiicola also showed slight but significant reductions in percentage germination and in germ-tube length, and considerable reductions in the proportion of germ-tubes which produced an appressorium, compared with spores on virus-free leaves.

Notes: A total of 404 isolates of Xanthomonas campestris pv. vesicatoria, obtained from Capsicum chinense cv. West Indian Red grown in Barbados and Grenada, were differentiated into pathogenic races, and of these, 96 were tested also for selected taxonomic group phenotypes. The response of C. chinense to infection by several X. campestris pv. vesicatoria races and the contribution of races isolated from this cultivar to severity of bacterial spot of bell pepper and tomato were also investigated. P4T2, P5T2 and P6T2 were the predominant races of X. campestris pv. vesicatoria isolated from C. chinense grown in Grenada, whereas nine races (T1, P4, P6, P0T2, P1T2, P4T1, P4T2, P6T1 and P6T2) were isolated in Barbados. Race P4T2 comprised 46.0 and 71.4% of the isolates from Barbados and Grenada, respectively. The 96 isolates, all of which overcame resistance conferred by the gene Bs2, shared taxonomic group B strain characteristics, including the presence of the β-protein band, positive amylolytic activity and inability to oxidize cis-aconitate. The C. chinense cv. West Indian Red was susceptible only to races of X. campestris pv. vesicatoria that can overcome Bs2 gene resistance. Of six such races identified in Barbados, only P4T1, P4T2 and P6T1 affected bacterial spot-susceptible bell pepper or tomato in the field, and they amounted to only 1.5–2.1% of each sample of isolates from these plant species. Moreover, they were confined to the smallest bacterial spot lesions. Bell pepper was most severely affected by combinations of races T1 with P3T2 and T2 with P0T1, and tomato by race T1 only and combinations of races P0T1 with P0T2 and P1T1 with P1T0, all of which prevailed in the field despite selection against them by C. chinense cv. West Indian Red.

Notes: Diversity in populations of Erysiphe graminis (Blumeria graminis) f.sp. hordei was studied with virulence and molecular markers. Isolates were sampled in two locations in northern France from a winter barley cultivar (Plaisant) and a spring cultivar (Caruso). Only a few pathotypes (determined by virulence markers) were common. The rest of the population was diverse. Diversity within common pathotypes, estimated by five RAPD and two SCAR markers, was generally high, except for one pathotype, which was frequent on Plaisant. This pathotype carried only one virulence, Va22, out of the 11 virulences tested. It appeared as a clonal lineage, which had occurred previously, at least in 1992, in northern France, demonstrating survival of asexual lineages in populations that often reproduce sexually.

Notes: The effects of abiotic variables on the response of carnation cultivars to Fusarium oxysporum f.sp. dianthi (F.o. dianthi ) were examined in experiments conducted under semi-controlled environments. The abiotic variables examined were solar radiation intensity, temperature and growth substrate. Temperature was not controlled, but differed markedly among experiments, thus, its effect was not determined quantitatively. Disease incidence and disease severity varied significantly among the experiments (due mainly to differences in temperature), among the solar radiation treatments and among the cultivars tested. The three-way interaction term (i.e. cultivar × shade treatment × experiment) was highly significant (P 〈 0.001) when both disease incidence and disease severity were considered, indicating that no single variable was predominant in determining disease intensity. The effects of the growth substrate on disease progress was examined in plants grown in tuff or in tuff mixed with peat (1 : 1 and 1 : 3) substrates. The growth substrate had a potent effect on disease development in the less susceptible cultivars. Severe epidemics developed in all cultivars when they were grown in the tuff/peat mixture, although some were resistant when grown in tuff alone. These results led to the conclusion that the carnation response to F.o. dianthi is substantially influenced by the environmental conditions of the test.

Notes: The symptoms of witches' broom disease in cocoa, caused by the Basidiomycete fungus Crinipellis perniciosa, are pronounced swelling of the terminal and axillary buds followed in the long term by necrosis of this tissue. The direct effect of C. perniciosa on cocoa cells was examined under controlled conditions by growing primary and secondary phase cultures of the fungus separately and also with callus cultures and with cell suspensions. Both primary and secondary phase mycelium reduced growth of callus cultures by about 47% after one week compared with the controls. However, cell suspensions containing primary phase mycelium showed initial growth double that of the uninfected controls after 5 days, but then growth was reduced below that of the control and particularly when the primary phase became secondary phase mycelium. This change in fungal development coincided with the time that the cell culture reached the stationary growth stage. Cell cultures inoculated with stationary phase mycelium showed the same growth as the control after 5 days but then growth was reduced to 50% of the control after 19 days incubation and remained at this low level subsequently. The inhibitory effect of secondary phase mycelium was examined by incubating callus and cell suspensions with culture filtrate from liquid cultures of the secondary phase. Inclusion of 50% by volume of culture filtrate from the secondary phase in the growth medium for callus and cell suspensions, respectively, resulted in a reduction in growth of the plant tissue cultures. Addition of fungal culture filtrates also led to loss in potassium and loss of viability of cell suspensions and of isolated cells as represented by protoplasts. The necrotrophic mode of the secondary phase may be achieved through the production of phytotoxins acting on the host cell membrane.

Notes: The random amplified polymorphic DNA (RAPD) technique was used to determine the mating groups of several members of the Fusarium section Liseola recovered from maize, rice and sorghum collected from different locations in Ghana. Three mating groups were identified, A, D and F, of which all A and F isolates were confirmed by mating studies. Fertile crosses were also obtained in crosses involving two of the isolates identified as belonging to the D population. Variability within the A population isolated from seeds and stem-bases of maize was investigated to determine whether the sub-structuring of this population was related to the host tissue from which the isolates were obtained. The relative merits of the RAPD procedure, compared to the mating procedure, for determining the mating affiliations of isolates and for more detailed analyses of isolates within a population, as well as its possible advantages over established RFLP methodologies are discussed.

Notes: To study the variation between wheat bymovirus isolates and to resolve uncertainties about the identity of the virus in some countries, leaves of infected plants were obtained from nine sites in China and from one each in Italy, Germany, USA and Canada. The German isolate was obtained from rye and the Canadian isolate was the type strain of wheat spindle streak mosaic virus (WSSMV). In RT-PCR, using primers designed from a partial sequence of a French isolate (tentatively described as WSSMV), genome fragments were obtained from the Italian and the French isolates but not from the Chinese ones. Conversely, products were consistently obtained from the Chinese isolates, but not from the Italian or French ones, when primers were designed from the sequence of a Japanese isolate of wheat yellow mosaic virus (WYMV). Nucleotide sequences were obtained from regions at or near the 3′-terminus of RNA1 of six Chinese isolates and the four from Europe and North America, usually including the coat protein. Nucleotide and amino acid sequence comparisons demonstrated that the European and North American isolates were extremely similar and were therefore WSSMV, while the Chinese isolates were close to the Japanese isolate and were thus WYMV.

Notes: Oilseed rape cultivars possess inadequate levels of resistance to light leaf spot disease, caused by the ascomycete Pyrenopeziza brassicae Sutton & Rawlinson. High levels of resistance to this disease were found within wild accessions of Brassica oleracea and B. rapa. This resistance was introgressed into agronomically acceptable winter oilseed rape breeding lines. Seedling resistance was determined by two genes. One of these, derived from B. rapa and positioned on linkage group N1, resulted in no apparent symptoms following infection, while the other, derived from B. oleracea and positioned on N16, resulted in black necrotic flecks and a reduced amount of sporulation compared with standard cultivars. Several agronomically acceptable double haploid lines were developed which expressed very high levels of adult plant resistance.

Notes: The dispersal of spores from lesions of brown (Puccinia recondita f.sp. tritici) or yellow (P. striiformis) rusts of wheat by impacting drops was studied. Using a generator of uniform-size drops, drops of 2.5, 3.4, 4.2 and 4.9 mm in diameter were released from rest at heights of 5, 50 and 100 cm above horizontal and primary leaves uniformly covered with sporulating lesions. Dry-dispersal and rain-splash occurred simultaneously in response to drop impaction. A coloration technique allowed separate counting of dry-dispersed and rain-splashed spores caught on slides. More spores were rain-splashed than dry-dispersed. Neither removal mechanism affected in-vitro germination of spores, which was higher in brown than in yellow rust. For both rusts, the number of both dry-dispersed and rain-splashed spores, as well as their travel distance, increased with drop diameter and fall height. The fall speed of incident drops in relation to diameter and fall height was obtained by solving numerically the equation of vertical drop motion. The number of spores removed by a given impacting drop was found to be a power function of the calculated kinetic energy of the impacting drop. Based on this experimental relationship, a simulation study showed the relevance of rain type in the removal of spores.

Notes: Six male sterile sunflower lines were crossed with seven restorers in a factorial mating design. The 13 inbred lines and their 42 F1 hybrids were planted in a randomized block design with three replicates. Each replicate consisted of two rows, 5 m long (30–35 plants per replicate). Resistance to natural Phomopsis infection, presented as the percentage of plants with no encircling necrosis lesions of the fungus on the main stem, was determined at physiological maturity. Analysis of variance showed that female and male general combining abilities (GCA) and specific combining abilities (SCA) of F1 hybrids were significant. The ratio of additive variance to total variance was 0.662, a high value which indicates prevailing additive effects. The additive variance due to females was more important than that of males, probably because of the existence of maternal effects or more effective genes for resistance in the female lines used in this experiment. The estimates of GCA were significant and positive for LC1004A, KO549A, 50KD8 and LC1064C inbred lines. These lines should be considered in developing hybrids with improved resistance to Phomopsis in sunflower breeding programmes.

Notes: Fenpropimorph-resistant mutants of Ustilago maydis were obtained at high frequency (30 × 10−6) after UV-irradiation followed by selection on media containing fenpropimorph (50 μg mL−1). Genetic analysis of 30 such mutants resulted in the identification of two unlinked chromosomal loci, the U/fpm-1 locus with two allelic genes (U/fpm-1A and U/fpm-1B) and the U/fpm-2 locus. The mutant genes U/fpm-1A and U/fpm-2 are responsible for high resistance levels (Rf: 75–100 or 257–286 based on MICs or ED50s, respectively), while the U/fpm-1B mutation gives only a small reduction (approximately 7–10-fold) in fenpropimorph sensitivity. Cross-resistance studies with other SBIs showed that the major gene (U/fpm-1A and U/fpm-2) mutants were cross-resistant to the related compound fenpropidin (Rf: 15–20 or 53–66 based on MICs or ED50s values, respectively) and to tridemorph (Rf: 5 or 7.1–9.5 based on MICs or ED50s values, respectively), but not to the inhibitors of steps of ergosterol biosynthesis preceding the Δ14-reductase. The minor gene (U/fpm-1B) mutants also had low-level resistance (approximately 5-fold) to tridemorph and to fenpropidin, but in contrast with the major gene mutants they were 2–10 times more sensitive to the triazoles studied (triadimefon, triadimenol, propiconazole and flusilazole) and to the pyridine, pyrifenox.Studies of the fitness of U. maydis mutants showed that in major gene mutants, resistance was not associated with changes in growth rate in liquid culture or pathogenicity on young maize plants. The minor gene mutation reduced significantly the growth rate in liquid culture and the pathogenicity, either in homozygous or heterozygous condition in dikaryotic mycelium.

Notes: Bacteria from necrotic branches of Asian pear trees (Pyrus pyrifolia) in Korea were consistently isolated as white colonies on nutrient agar and formed mucoid, slightly yellow colonies on a minimal medium with copper sulphate. Isolates with this colony morphology were studied in a series of microbiological, molecular and pathological tests. Most isolates allowed the verification of Koch's postulate on P. pyrifolia seedlings and on slices from immature pear (Pyrus communis) fruits and were also positive in hypersensitivity tests on tobacco leaves. They showed characteristics common to species in the genus Erwinia, but were different from Erwinia amylovora, the agent of fire blight. A relationship between the novel pathogen and E. amylovora was found in microbiological and serological tests. Both organisms had similar but not identical protein patterns in 2-D gel electrophoresis, and in growth morphology the new pathogen produced colonies on MM2 Cu medium that were mucoid and slightly yellow, compared with the clearly yellow colonies of E. amylovora. No similarity was found in the plasmid profiles, and consequently no PCR signal was obtained with primers from the E. amylovora plasmid pEA29. REP-PCR also produced bands differing for the two organisms.

Notes: The relative incidence of Erysiphe cichoracearum and Sphaerotheca fuliginea, both agents of powdery mildew of cucurbits, was determined from 275 samples of mildewed leaves of cucurbits collected in 1994 from five regions of France. E. cichoracearum was identified in 9 to 39% of the mildewed leaf samples from four of the regions but was not detected in samples from the Mediterranean island of Corsica. The genetic structure of the French population of E. cichoracearum was examined using RFLPs of the ribosomal internal transcribed spacers amplified by PCR, random amplified polymorphic DNA (RAPD) markers, pathogenicity and mating-type tests. Forty-one isolates, including one from England, were analysed. Cluster analysis from 147 RAPD fragments using 16 primers revealed the existence of three distinct genetic lineages corresponding to three rDNA haplotypes (designated groups A, B and C). Bootstrap, genetic diversity, gametic disequilibrium and private allele analyses supported this differentiation. The genetic differentiation observed in the French population was not related to the geographical origin of the isolates. Group A isolates may be more specialized on melon as, with one exception, they were of race 1 (growth on four of the five melon cultivars tested) in comparison with group B and C isolates, which were of race 0 (growth on IranH only). Thus, the genetic differentiation observed may indicate a host-specialized subdivision within the French population of E. cichoracearum from cucurbits. Gametic disequilibrium analysis among RAPD loci and biological observations suggest that the sexual stage is of minor importance for epidemics of E. cichoracearum on cucurbits.