Frank J. Louws

Development and Evaluation of PCR-Based Diagnostic Assays for the Bacterial Speck and Bacterial Spot Pathogens of Tomato

Bacterial speck and bacterial spot lesions can easily be confused with each other and with those formed by other tomato pathogens. To facilitate disease diagnosis, we developed and evaluated polymerase chain reaction (PCR)-based lesion assays using crude DNA extracts and primer sets COR1/2 (bacterial speck) and BSX1/2 (bacterial spot). All 29 pathogenic Pseudomonas syringae pv. tomato strains tested produced a 689-bp amplicon with COR1/2; 28 of the 37 geographically diverse bacterial spot-causing xanthomonad (BSX) strains that were tested generated the 579-bp BSX1/2 amplicon. The detection limit with plant samples was 30 to 50 CFU/reaction. In a 6-year study, the COR1/2 PCR assay diverged from the culture-based classical assay for only 3 of 70 bacterial speck lesion samples collected from Ontario greenhouses and tomato fields; the BSX1/2 assay was positive for 112 of the 124 confirmed bacterial spot lesions sampled. The majority (66%) of the BSX strains isolated from these lesions belonged to group D; the 12 strains that were BSX1/2-negative belonged to group C. Group D strains produced a 425-bp PCR product with crude DNA extracts but a 579-bp product with purified DNA; the former was identical to the latter except that it was missing 150 bp from the middle of the 579-bp sequence.

Grafting Tomato with Interspecific Rootstock to Manage Diseases Caused by Sclerotium rolfsii and Southern Root-Knot Nematode

Southern blight (Sclerotium rolfsii) and root-knot nematodes (Meloidogyne spp.) cause severe damage to fresh-market tomato (Solanum lycopersicum) throughout the southeastern United States. Grafting is an emerging technology in U.S. tomato production, and growers require information regarding the resistance characteristics conferred by rootstocks. In this study, southern blight (SB) and root-knot nematodes (RKN) were effectively managed using interspecific hybrid rootstocks. During 2007 and 2008, field trials were carried out at two locations that had soils naturally infested with S. rolfsii. At the end of the growing seasons, the mean SB incidence of nongrafted plants was 27 and 79% at the two sites. SB incidence among plants grafted onto rootstock cultivars Big Power (one location only), Beaufort, and Maxifort ranged from 0 to 5%, and area under the disease progress curve (AUDPC) values were lower than for nongrafted and self-grafted controls (P < 0.01). At one location, soils were naturally infested with RKN, and all three rootstocks reduced RKN AUDPC and RKN soil populations at first harvest (P < 0.01). Big Power was particularly effective at reducing RKN galling and RKN soil populations at final fruit harvest (P < 0.01). Fruit yield was higher when resistant rootstocks were utilized (P < 0.05), and in our study grafting was effective at maintaining crop productivity in soils infested with S. rolfsii and M. incognita.

CAN SIMULTANEOUS INHIBITION OF SEEDLING GROWTH AND STIMULATION OF RHIZOSPHERE BACTERIAL POPULATIONS PROVIDE EVIDENCE FOR PHYTOTOXIN TRANSFER FROM PLANT RESIDUES IN THE BULK SOIL TO THE RHIZOSPHERE OF SENSITIVE SPECIES

In order to demonstrate that allelopathic interactions are occurring, one must, among other things, demonstrate that putative phytotoxins move from plant residues on or in the soil, the source, through the bulk soil to the root surface, a sink, by way of the rhizosphere. We hypothesized that the incorporation of phytotoxic plant residues into the soil would result in a simultaneous inhibition of seedling growth and a stimulation of the rhizosphere bacterial community that could utilize the putative phytotoxins as a sole carbon source. If true and consistently expressed, such a relationship would provide a means of establishing the transfer of phytotoxins from residue in the soil to the rhizosphere of a sensitive species under field conditions. Presently, direct evidence for such transfer is lacking. To test this hypothesis, cucumber seedlings were grown in soil containing various concentrations of wheat or sunflower tissue. Both tissue types contain phenolic acids, which have been implicated as allelopathic phytotoxins. The level of phytotoxicity of the plant tissues was determined by the inhibition of pigweed seedling emergence and cucumber seedling leaf area expansion. The stimulation of cucumber seedling rhizosphere bacterial communities was determined by the plate dilution frequency technique using a medium containing phenolic acids as the sole carbon source. When sunflower tissue was incorporated into autoclaved (to reduce the initial microbial populations) soil, a simultaneous inhibition of cucumber seedling growth and stimulation of the community of phenolic acid utilizing rhizosphere bacteria occurred. Thus, it was possible to observe simultaneous inhibition of cucumber seedlings and stimulation of phenolic acid utilizing rhizosphere bacteria, and therefore provide indirect evidence of phenolic acid transfer from plant residues in the soil to the root surface. However, the simultaneous responses were not sufficiently consistent to be used as a field screening tool but were dependent upon the levels of phenolic acids and the bulk soil and rhizosphere microbial populations present in the soil. It is possible that this screening procedure may be useful for phytotoxins that are more unique than phenolic acids.

Polyphasic Characterization of Xanthomonas Strains from Onion

ABSTRACT Xanthomonas leaf blight has become an increasingly important disease of onion, but the diversity among Xanthomonas strains isolated from onion is unknown, as is their relationship to other species and pathovars of Xanthomonas. Forty-nine Xanthomonas strains isolated from onion over 27 years from 10 diverse geographic regions were characterized by pathogenicity to onion and dry bean, fatty acid profiles, substrate utilization patterns (Biolog), bactericide resistance, repetitive sequence-based polymerase chain reaction fingerprinting, rDNA internally transcribed spacer (ITS) region, and hrp b6 gene sequencing. Multiplication of onion Xanthomonas strain R-O177 was not different from X. axonopodis pv. phaseoli in dry bean, but typical common bacterial blight disease symptoms were absent in dry bean. Populations from each geographical region were uniformly sensitive to 100 mug of CuSO(4), 100 mug of ZnSO(4), and 100 mug of streptomycin sulfate per ml. Biolog substrate utilization and fatty acid profiles revealed close phenoltypic relatedness between onion strains of Xanthomonas and X. axonopodis pv. dieffenbachiae (57% of strains) and X. arboricola pv. poinsettiicola (37% of strains), respectively. A logistic regression model based on fatty acid composition and substrate utilization classified 69% of strains into their geographical region of origin. Sequencing of a portion of the hrp B6 gene from 24 strains and ITS region from 25 strains revealed greater than 97% sequence similarity among strains. DNA fingerprinting revealed five genotype groups within onion strains of Xanthomonas and a high degree of genetic diversity among geographical regions of origin. Based on pathogenicity to onion, carbon substrate utilization, fatty acid profiles, rDNA genetic diversity, and genomic fingerprints, we conclude that the strains examined in this study are pathovar X. axonopodis pv. allii. Implications of genetic and phenotypic diversity within X. axonopodis pv. allii are discussed in relation to an integrated pest management program.

Pathogenic and Genetic Relatedness Among Xanthomonas axonopodis pv. allii and Other Pathovars of X. axonopodis

ABSTRACT Xanthomonas axonopodis pv. allii is phenotypically and genetically diverse and its relationship to other X. axonopodis pathovars within DNA homology group 9.2 is unknown. In growth chamber experiments, disease symptoms were produced on onion only by inoculation with X. axonopodis pv. allii. Citrus bacterial spot symptoms were induced by X. axonopodis pvs. alfalfae, itrumelo, and allii on Duncan grapefruit and key lime. X. axonopodis pv. allii multiplication and persistence in Duncan grapefruit were equal to those of an aggressive strain of X. axonopodis pv. citrumelo, but populations of X. axonopodis pvs. alfalfae, betlicola, citrumelo, phaseoli, and vesicatoria were 1.3 to 4.0 log units less than X. axonopodis pv. allii in onion. Genomic fingerprinting by repetitive sequence- based polymerase chain reaction demonstrated that X. axonopodis pvs. allii, alfalfae, and citrumelo are distinct from other Xanthomonas species and X. axonopodis pathovars, but these pathovars were indistinguishable from each other. Three genotype groups were apparent among DNA homology group 9.2 strains, and generally correspond to the aggressiveness and genotype groups previously described for X. axonopodis pv. citrumelo. X. axonopodis pvs. allii, alfalfae, and citrumelo appear to have recently diverged from a common ancestral strain.

Impact of reduced fungicide and tillage on foliar blight, fruit rot, and yield of processing tomatoes

The effect of reduced tillage, soil-surface crop residue maintenance, and reduced fungicide input on processing tomato yield and disease incidence was studied in 1990 to 1992. Fall-seeded rye was desiccated in strips in early spring; the remainder, after 1.2 m of growth. Strips were zone tilled (ZT) 35 cm deep with no soil inversion. The ZT system permitted desiccated interrow rye residue to persist throughout the summer, providing approximately 90% cover of the soil surface. Tomatoes were transplanted into the prepared strips. The ZT system did not affect marketable yield or percent fruit with mold (1991 to 1992); but it decreased (1990), increased (1991), and did not affect (1992) defoliation caused by early blight (EB) compared to a conventional tillage production system using a moldboard plow, disk, or both. The fungicide, Bravo 720 (chlorothalonil), was applied as follows: none, weekly, or a full or reduced rate at intervals according to the disease forecasting model, TOM-CAST. Fungicide treatment did not enhance marketable yield compared to that of the unsprayed treatment. TOM-CAST-based treatments did not consistently provide control of defoliation compared to that in plots sprayed weekly. However, compared to weekly sprays, select forecast-generated spray schedules required 45 to 80% fewer applications to limit fruit mold incidence caused by Alternaria solani (EB), Colletotrichum coccodes (anthracnose), and Rhizoctonia solani (soil rot). Conservation tillage practices, soil-surface residue maintenance, and reduced fungicide input were integrated without compromising yield and management of disease, affording advantages of sustained farmland productivity.

Differential response of tomato genotypes to Xanthomonas-specific pathogen-associated molecular patterns and correlation with bacterial spot (Xanthomonas perforans) resistance.

Plants depend on innate immune responses to retard the initial spread of pathogens entering through stomata, hydathodes or injuries. These responses are triggered by conserved patterns in pathogen-encoded molecules known as pathogen-associated molecular patterns (PAMPs). Production of reactive oxygen species (ROS) is one of the first responses, and the resulting ‘oxidative burst’ is considered to be a first line of defense. In this study, we conducted association analyses between ROS production and bacterial spot (BS; Xanthomonas spp.) resistance in 63 genotypes of tomato (Solanum lycopersicum L.). A luminol-based assay was performed on leaf tissues that had been treated with a flagellin 22 (flg22), flagellin 28 and a Xanthomonas-specific flg22 (flg22-Xac) peptide, to measure PAMP-induced ROS production in each genotype. These genotypes were also assessed for BS disease response by inoculation with Xanthomonas perforans, race T4. Although there was no consistent relationship between peptides used and host response to the BS, there was a significant negative correlation (r=−0.25, P<0.05) between foliar disease severity and ROS production, when flg22-Xac was used. This response could potentially be used to identify the Xanthomonas-specific PRR allele in tomato, and eventually PAMP-triggered immunity loci could be mapped in a segregating population. This has potential significance in tomato improvement.

Resistance to Xanthomonas perforans race T4 causing bacterial spot in tomato breeding lines

&NA; Tomato (Solanum lycopersicum) is the second most important vegetable crop in the world. Bacterial spot (BS) of tomato, caused by four species of Xanthomonas: X. euvesicatoria, X. vesicatoria, X. perforans and X. gardneri, results in severe loss in yield and quality due to defoliation and formation of lesions on fruits, respectively. Currently management practices do not offer effective control under conditions of high disease pressure. Thus, developing BS resistance is a critical priority for tomato growers in order to minimize crop losses. Sixty‐three advanced tomato breeding lines, heirlooms and wild tomato lines with diverse genetic backgrounds were screened under greenhouse and field conditions for BS resistance using X. perforans race T4, which was found to be a prevalent race in North Carolina. Race T4 isolate 9 was used to inoculate the plants by spraying, and disease severity was measured using the Horsfall‐Barratt scale. Tomato lines 74L‐1W(2008), NC2CELBR, 081–12–1X‐gsms, NC22L‐1 (2008) and 52LB‐1 showed resistance to BS in the field and/or greenhouse trials. These lines were derived from S. pimpinellifolium L3707. Screening L3707 followed by development of a mapping population and mapping resistance genes might be useful for breeding resistance against BS in future breeding programmes.

Rhizoctonia communities in soybean fields and their relation with other microbes and nematode communities

Soybean root rot, caused by Rhizoctonia solani, is one of the most serious soybean diseases in the North Central Region of the United States. There is no report of the relationship between Rhizoctonia root rot and soil physical and chemical properties, or microbial and nematode communities. A commercial soybean field with a long history of Rhizoctonia root rot was examined to explore this relationship. Results demonstrated that high disease incidence in sampled areas was positively correlated with high levels of nitrogen, phosphorus, manganese, populations of soil Rhizoctonia, thermophiles and fungi, root Rhizoctonia colonies, populations of lesion and stunt nematodes, and negatively correlated with high levels of calcium, magnesium, sodium, base saturation, and populations of Pseudomonas based on correlation analysis and canonical correspondence analysis (CCA). Cluster analysis showed that the communities of both Rhizoctonia and bacteria in sampled areas were separated based on healthy and diseased plants using denaturing gradient gel electrophoresis (DGGE); however, there was no clear separation for Fusarium, Pythium and Trichoderma communities in sampled areas based on healthy and diseased plants, indicating these species might not be directly associated with Rhizoctonia root rot. Moreover, the disease suppression seems to be more related to the quantity of soil beneficial microorganisms rather than specific species. In addition, DGGE is a reliable technique to characterize microbial communities and identify fungal and bacterial species in complex soil systems.

IPM for Soilborne Disease Management for Vegetable and Strawberry Crops in SE USA

Major shifts in agricultural practices are complex with highly inter-dependent biological, environmental, social, economic, business, and other (agri)culture factors. Seeking alternatives to methyl bromide (MeBr) to manage soilborne pathogens provided a model system that could be simulated where other large scale and dramatic changes need to be made. Parallel priorities were set in place. The first priority was to assemble an inter-disciplinary and inter-state response and vision team of key private and public sector stakeholders. The second priority was to determine the risk-aversion and biological basis for fumigation. The third priority was to implement a plan that did not simply focus on chemical alternatives, but sought to advance the science of plant pathology and conduct discovery research about the biology, ecology and management of the primary plant pathogens and cropping systems. Therefore, three strategic levels of research and extension were identified: (1) Tactic substitution – addressing short term needs of growers who sought non-ozone depleting fumigant alternatives; (2) Tactic Diversification – focused on medium term alternatives that included non-fumigant and IPM based tactics; (3) Tactic Development – focused on long-term goals to explore microbial ecology and farming systems-based approaches to replace MeBr-dependent production systems. The fourth priority was to effectively extend research based information to primary clientele. Combined efforts resulted in technically and economical feasibility assessments of alternatives, exploration of viable diversification and development of plant disease management tactics, and a region-wide advanced understanding of the biology and ecology of key plant pathogens.