Detection and characterization of broad-spectrum antipathogen activity of novel rhizobacterial isolates and suppression of Fusarium crown and root rot disease of tomato.

AIMS: To detect and characterize broad-spectrum antipathogen activity of indigenous bacterial isolates obtained from potato soil and soya bean leaves for their potential to be developed as biofungicides to control soilborne diseases such as Fusarium crown and root rot of tomato (FCRR) caused by Fusarium oxysporum f. sp. radicis-lycopersici (Forl). METHODS AND RESULTS: Thirteen bacterial isolates (Bacillus amyloliquefaciens (four isolates), Paenibacillus polymyxa (three isolates), Pseudomonas chlororaphis (two isolates), Pseudomonas fluorescens (two isolates), Bacillus subtilis (one isolate) and Pseudomonas sp. (one isolate)) or their volatiles showed antagonistic activity against most of the 10 plant pathogens in plate assays. Cell-free culture filtrates (CF) of five isolates or 1-butanol extracts of CFs also inhibited the growth of most pathogen mycelia in plate assays. PCR analysis confirmed the presence of most antibiotic biosynthetic genes such as phlD, phzFA, prnD and pltC in most Pseudomonas isolates and bmyB, bacA, ituD, srfAA and fenD in most Bacillus isolates. These bacterial isolates varied in the production of hydrogen cyanide (HCN), siderophores, ß-1,3-glucanases, chitinases, proteases, indole-3-acetic acid, salicylic acid, and for nitrogen fixation and phosphate solubilization. Gas chromatography-mass spectrometry analysis identified 10 volatile compounds from 10 isolates and 18 compounds from 1-butanol extracts of CFs of five isolates. Application of irradiated peat formulation of six isolates to tomato roots prior to transplanting in a Forl-infested potting mix and field soil provided protection of tomato plants from FCRR disease and enhanced plant growth under greenhouse conditions. CONCLUSIONS: Five of the 13 indigenous bacterial isolates were antagonistic to eight plant pathogens, both in vitro and in vivo. Antagonistic and plant-growth promotion activities of these isolates might be related to the production of several types of antibiotics, lytic enzymes, phytohormones, secondary metabolites, siderophores and volatile compounds; however, any specific role of each needs to be determined. SIGNIFICANCE AND IMPACT OF THE STUDY: Indigenous antagonistic bacterial isolates have the potential to be developed as biofungicides for minimizing early crop losses due to soilborne diseases caused by Fusarium and other soilborne pathogens.

Characterization of Chinese eggplant isolates of the fungal pathogen Verticillium dahliae from different geographic origins.

Verticillium dahliae is a fungal pathogen that causes wilt disease in a wide range of host plants. Characterization of virulence, morphological, and molecular variations among V. dahliae isolates from different geographic origins is essential for any breeding program aimed at producing plant cultivars resistant to this disease. We characterized virulence variation among V. dahliae isolates from Chinese cultivated eggplant grown in Northeast China by pathogenicity testing on susceptible, moderately resistant, and resistant eggplant accessions in a glasshouse using a root-dipping method of infection. These isolates were also characterized for morphological features based on growth on potato dextrose agar media and for genetic variation based on ISSR markers. All 12 isolates were divided into three pathotypes based on the virulence, i.e., strong, moderate, or weak type. Three isolates were categorized as defoliating pathotypes, with strong virulence, and the rest of the isolates were categorized as non-defoliating pathotypes, with moderate to weak virulence. The eggplant isolates were classified into three morphological types or morphotypes, hypha, hypha-sclerotia, and sclerotia; no significant correlations were detected between pathotypes and morphotypes or geographic origins. ISSR fingerprinting indicated genetic diversity among isolates, ranging from 0.26 to 0.69. Specific fingerprint types were not correlated with either pathotype or morphotype. However, ISSR analyses did reveal two clusters in which the isolates in each share the same or neighboring geographic origins.

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.

Diversity Among Strains of Xanthomonas campestris pv. vitians from Lettuce.

ABSTRACT Diversity in host range, pathogenicity, phenotypic characteristics, repetitive extragenic palindromic polymerase chain reaction (rep-PCR) profiles, and sequence of the 16S-23S rDNA spacer region was examined among 44 Xanthomonas strains isolated from lettuce. Forty-two of the strains were divided into two groups, designated A and B. Seventy percent were Group A, and most of the remaining strains including a reference strain (LMG 938) were Group B. Group A strains induced both local and systemic symptoms, whereas Group B strains caused only distinct necrotic spots. Two strains, including the X. campestris pv. vitians type strain, were distinct from the Group A and B strains and were not pathogenic on lettuce. Analysis of fatty acid profiles, serotype, carbon substrate utilization patterns, and protein fingerprints confirmed this grouping. The Group A and B strains also formed two unique clusters (I and II) by rep-PCR profiling that corresponded to the two groups. Direct sequencing of a PCR-amplified DNA fragment (680 bp) from the 16S-23S rDNA spacer region of four representative strains, however, did not differentiate these groups. Serology and rep-PCR fingerprinting can be used to diagnose and identify X. campestris pv. vitians strains, while the other analyses evaluated are useful for strain characterization.

Effect of Compost Amendments on Disease Severity and Yield of Tomato in Conventional and Organic Production Systems.

Field trials were conducted over 2 years to assess the effects of compost amendments on disease development in organic and conventional processing tomato (Lycopersicon esculentum L.) production systems. The incidence of anthracnose fruit rot was reduced in organic tomato plots amended with a high rate of composted cannery wastes compared with the incidence in nonamended control plots in 1998 when disease incidence was high. Marketable yield was increased by 33% in compost-amended organic plots. Plots amended with a high compost rate had more ripe fruit than the nonamended control. The incidence of anthracnose and of total disease on fruit was less on the cultivar OH 8245 than on Peto 696. Total fruit yield of OH 8245 but not Peto 696 in organic plots was increased by amendment with composted cannery wastes. In conventional tomato production, composted yard wastes increased disease severity on foliage both years but reduced bacterial spot incidence on fruit in 1997, when disease pressure was high. The incidence of anthracnose was not affected by composted yard wastes. Marketable and total fruit yields of Peto 696 were not increased in compost-amended conventional plots. The plant activator Actigard reduced foliar disease severity and the incidence of bacterial spot and anthracnose on fruit, while increasing yield of marketable fruit.

Precise detection and tracing of Trichoderma hamatum 382 in compost-amended potting mixes by using molecular markers.

Randomly amplified polymorphic DNA (RAPD) analysis and the PCR assay were used in combination with dilution plating on a semiselective medium to detect and enumerate propagules of Trichoderma hamatum 382, a biocontrol agent utilized in compost-amended mixes. Distinct and reproducible fingerprints were obtained upon amplification of purified genomic DNA of T. hamatum 382 with the random primers OPE-16, OPH-19, and OPH-20. Three amplified DNA fragments of 0.35 (OPE-16(0.35)), 0.6 (OPH-19(0.6)), and 0.65 (OPH-20(0.65)) kb were diagnostic for T. hamatum 382, clearly distinguishing it from 53 isolates of four other Trichoderma spp. tested. Some isolates of T. hamatum shared these low-molecular-weight fragments with T. hamatum 382. However, RAPD analysis of isolates of T. hamatum with all three random primers used in consecutive PCR tests distinguished T. hamatum 382 from other isolates of T. hamatum. These three RAPD amplicons were cloned and sequenced, and pairs of oligonucleotide primers for each cloned fragment were designed. Use of the primers in the PCR assay resulted in the amplification of DNA fragments of the same size as the cloned RAPD fragments from genomic DNA of T. hamatum 382. A combination of dilution plating on a semiselective medium for Trichoderma spp. and PCR, with the RAPD primers OPH-19, OPE-16, and OPH-20 or the three sequence-characterized primers, was used successfully to verify the presence of T. hamatum 382 propagules in nine different soil, compost, and potting mix samples. All 23 Trichoderma isolates recovered on semiselective medium from commercial potting mixes fortified with T. hamatum 382 were identified as T. hamatum 382, whereas 274 Trichoderma isolates recovered from the other nine samples were negative in the PCR assay. Thus, this highly specific combination of techniques allowed detection and enumeration of propagules of T. hamatum 382 in fortified compost-amended potting mixes. Sequence-characterized amplified region markers also facilitated the development of a very simple procedure to amplify DNA of T. hamatum 382 directly from fortified compost-amended potting mixes.