Effect of acidic electrolyzed water on the viability of bacterial and fungal plant pathogens and on bacterial spot disease of tomato.

Acidic electrolyzed water (AEW), known to have germicidal activity, was obtained after electrolysis of 0.045% aqueous solution of sodium chloride. Freshly prepared AEW (pH 2.3-2.6, oxidation-reduction potential 1007-1025 mV, and free active chlorine concentration 27-35 ppm) was tested in vitro and (or) on tomato foliage and seed surfaces for its effects on the viability of plant pathogen propagules that could be potential seed contaminants. Foliar sprays of AEW were tested against bacterial spot disease of tomato under greenhouse and field conditions. The viability of propagules of Xanthomonas campestris pv. vesicatoria (bacterial spot pathogen), Streptomyces scabies (potato scab pathogen), and Fusarium oxysporum f.sp. lycopersici (root rot pathogen) was significantly reduced 4-8 log units within 2 min of exposure to AEW. Immersion of tomato seed from infected fruit in AEW for 1 and 3 min significantly reduced the populations of X. campestris pv. vesicatoria from the surface of the seed without affecting seed germination. Foliar sprays of AEW reduced X. campestris pv. vesicatoria populations and leaf spot severity on tomato foliage in the greenhouse. In the field, multiple sprays of AEW consistently reduced bacterial spot severity on tomato foliage. Disease incidence and severity was also reduced on fruit, but only in 2003. Fruit yield was either enhanced or not affected by the AEW sprays. These results indicate a potential use of AEW as a seed surface disinfectant or contact bactericide.

Effect of Soil Application of AG3 Phosphonate on the Severity of Clubroot of Bok Choy and Cabbage Caused by Plasmodiophora brassicae.

Field trials were carried out to test the effect of phosphonate fungicide (AG3) on the severity of clubroot of bok choy (Brassica rapa var. chinensis) and cabbage (B. rapa var. perkinensis and B. oleracea var. capitata) in commercial Ontario muck fields with a clubroot history. Disease severity also was examined in the same infested soil under greenhouse and microplot conditions. In microplot trials with bok choy, AG3 phosphonate concentrations of 0.07 and 0.14% a.i. applied before or after planting consistently reduced clubroot severity (1-to-4 rating) by 0.8 to 1.6 when planted in May or June. However, only the 0.14% a.i. preplanting treatment was effective in trials in July and August. Postplanting drenches of 0.14% a.i. were consistently effective throughout the season. Fresh weight of bok choy was increased or not affected by phosphonate treatments. Under field conditions, one (0.07, 0.14, and 0.21% a.i.) or two (0.07% a.i.) postplant-ing drench applications of phosphonate significantly reduced the incidence of clubroot by 52 to 87% and severity by 1.7 to 2.5 on bok choy in 2004 but not in 2005. In the 2004 trial, two applications of 0.07% a.i. AG3 phosphonate reduced the severity of clubroot comparably to single applications at 0.14 and 0.21% a.i. rates. Fresh weight of bok choy was increased by 34 to 86% with all phosphonate drench treatments in both years. With cabbage, AG3 postplanting drench treatments consistently reduced the severity of clubroot (1-to-5 rating) by a range of 0.7 to 3.3 under greenhouse, microplot, and field conditions. In the greenhouse, a single drench application of 0.07 and 0.14% a.i. AG3 phosphonate 1 day after transplanting cabbage seedlings to the infested muck soil reduced clubroot severity by 2.6 to 3.3 and increased fresh weight of cabbage tops by 66 to 69%. Similar results were seen with cabbage trials under both microplot and field conditions at all AG3 postplanting drench concentrations.

Seed Treatment with Phosphonate (AG3) Suppresses Pythium Damping-off of Cucumber Seedlings.

A formulation of phosphonate (AG3) was tested as a seed treatment for the control of Pythium damping-off of cucumber (Cucumis sativus L.) plants under controlled environment and field conditions. Cucumber seed were treated by soaking for 10 min in phosphonate solution. They were then planted into peat-based mix or sandy-loam soil mixed with Pythium aphanidermatum or P. ultimum inoculum or into muck soil naturally infested with P. irregulare, P. ultimum, and other Pythium spp. Under growth-room conditions, phosphonate seed treatment provided more than 80% control of damping-off in all infested substrates tested. Effective disease control was obtained even when treated seed were stored for 5 weeks and up to 18 months prior to planting. In microplots containing naturally infested muck soil, phosphonate seed treatment decreased the percentage of diseased cucumber plants and increased total fresh weights compared with untreated seed and phosphonate post-planting drench. In field-plot tests 6 weeks after planting treated seed in Pythium-infested muck soil, cucumber stands were 63% compared with 18% in the control, which had no phosphonate exposure, and 53% in the post-planting drench. Tests for potential phytotoxicity in the greenhouse showed that radish and bok choy germination was reduced by phosphonate treatment but corn, cucumber, soybean, sugar beet, tomato, and wheat were not affected. Phosphonate seed treatment is a cost-effective way of protecting cucumber plants from Pythium damping-off.

1,8-Dihydroxynaphthalene monoglucoside, a new metabolite of Sclerotinia sclerotiorum, and the effect of tricyclazole on its production.

Isolate SS7 of Sclerotinia sclerotiorum was previously shown to produce and excrete into agar medium copious amounts of the melanin precursor 1,8-dihydroxynaphthalene. Much reduced quantities of this product were produced in the presence of tricyclazole, an inhibitor of pentaketide melanin biosynthesis. In this study, we demonstrate that young cultures of isolate SS7 produce 1,8-dihydroxynaphthalene monoglucoside, a new natural product not previously reported from fungi. When cultured in the presence of tricyclazole, such young cultures also accumulated two new monoglucosides of 1,3,8-trihydroxynaphthalene, which, as well as 1,8-dihydroxynaphthalene monoglucoside, were also obtained from cultures of two other isolates of S. sclerotiorum. It is proposed that rapid glucosylation of 1,3,8-trihydroxynaphthalene in young tricyclazole-inhibited S. sclerotiorum cultures accounts for the failure to observe 2-hydroxyjuglone or other metabolites usually associated with blockage of the pentaketide pathway to melanin in fungi.

Differential Colonization of Tomato Roots by Nonpathogenic and Pathogenic Fusarium oxysporum Strains May Influence Fusarium Wilt Control.

ABSTRACT Histochemical staining, beta-glucuronidase (GUS) activity, or placing roots on agar were methods used to characterize interactions between the pathogenic fungus, Fusarium oxysporum f. sp. lycopersici, and the nonpathogenic biocontrol F. oxysporum strain 70T01 with respect to colonization behaviors, interaction sites, and population densities on tomato roots. Mycelia of strain 70T01, a genetic transformant expressing stable GUS activity, hygromycin B resistance, and effective disease control, were localized in epidermal and cortex cell layers of tomato roots in a discontinuous and uneven pattern. In contrast, mycelia of F. oxysporum f. sp. lycopersici were found in the vascular bundles. Thus, direct interactions between the two fungi likely happen in the root surface cell layers. Colonization density of strain 70T01 was related to the inoculation density but decreased with distance from the inoculation site. Host defense reactions, including increased cell wall thickness or papilla deposits, were adjacent to 70T01 hyphae. Experiments done in soil showed that strain 70T01 densities in roots were highest at inoculation zones and barely detectable for root segments more than 2 cm away from the inoculation sites. F. oxysporum f. sp. lycopersici densities were lowest at 70T01 inoculation zones and highest (>10 times) where strain 70T01 was not directly applied. Newly elongating roots where strain 70T01 did not reach were available for infection by the pathogen. The higher strain 70T01 density was always found when the plants were simultaneously infected by F. oxysporum f. sp. lycopersici, suggesting that F. oxysporum f. sp. lycopersici has as much influence in predisposing the plant to colonization by strain 70T01 as strain 70T01 has on providing disease protection against the pathogen.

Interlaboratory comparison of methods To quantify microsclerotia of verticillium dahliae in soil

In a comparison of different methods for estimating Verticillium dahliae in soil, 14 soil samples were analyzed in a blinded fashion by 13 research groups in seven countries, using their preferred methods. One group analyzed only four samples. Twelve soil samples were naturally infested, and two had known numbers of microsclerotia of V. dahliae added to them. In addition, a control was included to determine whether transport had an effect on the results. Results differed considerably among the research groups. There was a 118-fold difference between the groups with the lowest and highest mean estimates. Results of the other groups were evenly distributed between these extremes. In general, methods based on plating dry soil samples gave higher numbers of V. dahliae than did plating of an aqueous soil suspension. Recovery of V. dahliae from samples with added microsclerotia varied from 0 to 59%. Most of the variability within each analysis was at the petri dish level. The results indicate the necessity to check the performance of detection assays regularly by comparing recoveries with other laboratories, using a common set of soil samples. We conclude that wet plating assays are less accurate than dry plating assays.

Production of an extracellular trypsin-like protease by the fungal plant pathogen Verticillium dahliae.

The plant pathogenic fungus Verticillium dahliae produced extracellular alkaline protease activity when grown in liquid medium supplemented with a protein source. A serine protease was purified 80-fold in a single step, using cation-exchange chromatography, from the filtrate of cultures grown with skim milk as a protein source. N-terminal amino acid sequence analysis of the 30-kDa protein (VDP30) that copurified with the serine protease activity suggested that VDP30 is a trypsin-like protein. The purified enzyme hydrolyzed the synthetic substrate N alpha-benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA), and the activity on BAPNA was inhibited by leupeptin, further verifying the trypsin-like nature of the enzyme.

Growth Enhancement and Developmental Modifications of in Vitro Grown Potato (Solanum tuberosum spp. tuberosum) as Affected by a Nonfluorescent Pseudomonas sp.

A plant growth-promoting rhizobacterium, designated Ps JN and isolated from onion roots, was identified as a nonfluorescent Pseudomonas sp. The percentage of similarity of Ps JN to P. gladioli (NCPPB 1891), P. cichorii (NCPPB 943), and P. viridiflava (NCPPB 635), as determined from 135 biochemical and physiological tests was 77, 70, and 66%, respectively. Ps JN persisted through successive generations of in vitro cultured potato plantlets, both as endophytic and epiphytic populations. In vitro inoculated potato (Solanum tuberosum) nodal explants produced plantlets with significant increases in root number (24-196%), root dry weight (44-201%), haulm dry weight (14-151%), and stem length (26-28%) as compared with noninoculated control plants. Bacterization also enhanced leaf hair formation (55-110%), secondary root branching, and total plant lignin content (43%). Other root colonizing bacteria or heat-killed cells of Ps JN had no significant effect on plant growth. Detached leaves from in vitro grown control plants, when exposed to 19 degrees C and 50% relative humidity, lost 55% of their moisture content in 2.5 hours. Moisture loss by leaves of in vitro grown, bacterized plants, as well as greenhouse-acclimated, bacterized plants, and control plants, was less than 20%. Changes in stomatal closure appear to account for this difference.

Enzyme-linked immunosorbent assay on nitrocellulose membranes (dot-ELISA) in the serodiagnosis of plant pathogenic bacteria.

The usefulness of enzyme-linked immunosorbent assay on nitrocellulose membranes (dot-ELISA) for diagnosis and identification of plant pathogenic bacteria was tested. Five pathovars of Xanthomonas campestris and two antisera, one produced against pv. vesicatoria and the other against pv. translucens, were used in a model system. A 10-min incubation of the bacterial cells, dot blotted on membranes, in diluted sera, followed by either alkaline phosphatase conjugated protein A or goat antirabbit globulin, resulted in a specific reaction between the homologous serum and bacteria. Populations of 1000-2000 cfu per spot (ca. 0.3 cm2) could be detected with these reagents. The streptavidin-biotinylated peroxidase complex produced a definitive reaction with as few as 800 cfu, but cross-reactions became evident at the higher cell concentrations among all five pathovars in tests with both antisera. Cell-free extracts, obtained by centrifugation of boiled bacteria, reacted similarly to live cells. Unrelated bacteria did not react with either antiserum. Extracts of lesions from tomato and pepper leaves infected with X. campestris pv. vesicatoria reacted positively with the antiserum produced against this pathovar but not that produced with pv. translucens. Samples of supernatants from boiled lesions reacted with similar intensity as those from homogenized tissues.