Distribution of SUN, OVATE, LC, and FAS in the tomato germplasm and the relationship to fruit shape diversity.

Phenotypic diversity within cultivated tomato (Solanum lycopersicum) is particularly evident for fruit shape and size. Four genes that control tomato fruit shape have been cloned. SUN and OVATE control elongated shape whereas FASCIATED (FAS) and LOCULE NUMBER (LC) control fruit locule number and flat shape. We investigated the distribution of the fruit shape alleles in the tomato germplasm and evaluated their contribution to morphology in a diverse collection of 368 predominantly tomato and tomato var. cerasiforme accessions. Fruits were visually classified into eight shape categories that were supported by objective measurements obtained from image analysis using the Tomato Analyzer software. The allele distribution of SUN, OVATE, LC, and FAS in all accessions was strongly associated with fruit shape classification. We also genotyped 116 representative accessions with additional 25 markers distributed evenly across the genome. Through a model-based clustering we demonstrated that shape categories, germplasm classes, and the shape genes were nonrandomly distributed among five genetic clusters (P < 0.001), implying that selection for fruit shape genes was critical to subpopulation differentiation within cultivated tomato. Our data suggested that the LC, FAS, and SUN mutations arose in the same ancestral population while the OVATE mutation arose in a separate lineage. Furthermore, LC, OVATE, and FAS mutations may have arisen prior to domestication or early during the selection of cultivated tomato whereas the SUN mutation appeared to be a postdomestication event arising in Europe.

Linking sequence to function in soil bacteria: sequence-directed isolation of novel bacteria contributing to soilborne plant disease suppression.

Microbial community profiling of samples differing in a specific ecological function, i.e., soilborne plant disease suppression, can be used to mark, recover, and ultimately identify the bacteria responsible for that specific function. Previously, several terminal restriction fragments (TRF) of 16S rRNA genes were statistically associated with damping-off disease suppression. This work presents the development of sequence-based TRF length polymorphism (T-RFLP)-derived molecular markers to direct the identification and isolation of novel bacteria involved in damping-off pathogen suppression. Multiple sequences matching TRF M139 and M141 were cloned and displayed identity to multiple database entries in the genera incertae sedis of the Burkholderiales. Sequences matching TRF M148, in contrast, displayed greater sequence diversity. A sequence-directed culturing strategy was developed using M139- and M141-derived markers and media reported to be selective for the genera identified within this group. Using this approach, we isolated and identified novel Mitsuaria and Burkholderia species with high levels of sequence similarity to the targeted M139 and M141 TRF, respectively. As predicted, these Mitsuaria and Burkholderia isolates displayed the targeted function by reducing fungal and oomycete plant pathogen growth in vitro and reducing disease severity in infected tomato and soybean seedlings. This work represents the first successful example of the use of T-RFLP-derived markers to direct the isolation of microbes with pathogen-suppressing activities, and it establishes the power of low-cost molecular screening to identify and direct the recovery of functionally important microbes, such as these novel biocontrol strains.

Field management effects on damping-off and early season vigor of crops in a transitional organic cropping system.

Transitioning farmland to certified organic vegetable production can take many paths, each varying in their costs and benefits. Here, the effects of four organic transition strategies (i.e., tilled fallowing, mixed-species hay, low-intensity vegetables, and intensive vegetable production under high tunnels), each with and without annual compost applications for 3 years prior to assessment, were characterized. Although transition cropping strategies differed in soil chemistry (P < 0.05), the magnitude of the changes typically were marginal and pairwise comparisons were rarely significant. In contrast, the compost amendment had a much greater impact on soil chemistry regardless of cropping strategy. For example, percent C and total P increased by 2- to 5-fold and K increased from 6- to 12-fold. Under controlled conditions, damping-off of both edamame soybean (cv. Sayamusume) and tomato (cv. Tiny Tim) was reduced from 2 to 30% in soils from the mixed-hay transition. In the field, damping-off of both crops was also significantly lower in plots previously cropped to hay (P < 0.05). Although not always significant (P < 0.05), this pattern of suppression was observed in all four of the soybean experiments and three of the four tomato experiments independent of compost application. The compost amendments alone did not consistently suppress damping-off. However, plant height, fresh weight, and leaf area index of the surviving seedlings of both crops were greater in the compost-amended soils regardless of the transitional cropping treatment used (P < 0.05 for most comparisons). These data indicate that mixed-hay cropping during the transition periods can enhance soil suppressiveness to damping-off. In addition, although compost amendments applied during transition can improve crop vigor by significantly enhancing soil fertility, their effects on soilborne diseases are not yet predictable when transitioning to certified organic production.

2,4-diacetylphloroglucinol alters plant root development.

Pseudomonas fluorescens isolates containing the phlD gene can protect crops from root pathogens, at least in part through production of the antibiotic 2,4-diacetylphloroglucinol (DAPG). However, the action mechanisms of DAPG are not fully understood, and effects of this antibiotic on host root systems have not been characterized in detail. DAPG inhibited primary root growth and stimulated lateral root production in tomato seedlings. Roots of the auxin-resistant diageotropica mutant of tomato demonstrated reduced DAPG sensitivity with regards to inhibition of primary root growth and induction of root branching. Additionally, applications of exogenous DAPG, at concentrations previously found in the rhizosphere of plants inoculated with DAPG-producing pseudomonads, inhibited the activation of an auxin-inducible GH3 promoter::luciferase reporter gene construct in transgenic tobacco hypocotyls. In this model system, supernatants of 17 phlD+ P. fluorescens isolates had inhibitory effects on luciferase activity similar to synthetic DAPG. In addition, a phlD() mutant strain, unable to produce DAPG, demonstrated delayed inhibitory effects compared with the parent wild-type strain. These results indicate that DAPG can alter crop root architecture by interacting with an auxin-dependent signaling pathway.

Diversity of Ralstonia solanacearum Infecting Eggplant in the Philippines.

ABSTRACT The diversity of Ralstonia solanacearum strains isolated from eggplant (Solanum melongena) grown in five provinces of the Philippine island group of Luzon was assessed using a recently described hierarchical system. All strains keyed to race 1, biovar 3 or 4. Phylotype-specific multiplex polymerase chain reaction (PCR) indicated that, like most other strains of Asian origin, all the strains in our Philippine collection belong to phylotype I. Taxometric and phylogenetic analyses of partial endoglucanase gene sequences of strains from this collection and those previously deposited into GenBank revealed at least four subgroups among the otherwise monophyletic phylotype I strains. Nucleotide polymorphisms within each subgroup were infrequent and, among the subgroups identified in this study, variation was always <1.3%, indicating that the large majority of strains could be assigned to a single sequevar. Genomic DNA fingerprinting using enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed additional fine-scale genetic variation that was consistent with the endogluconase sequence data. Whole-pattern and band-based analyses of the genomic fingerprint data revealed four and eight distinct genotypes, respectively, within our collection. Eggplant from infested fields in different provinces tended to harbor mixed populations of ERIC genotypes, with the predominant genotype varying by location.

Farm Management Effects on Rhizosphere Colonization by Native Populations of 2,4-Diacetylphloroglucinol-Producing Pseudomonas spp. and Their Contributions to Crop Health.

ABSTRACT Analyses of multiple field experiments indicated that the incidence and relative abundance of root-colonizing phlD+ Pseudomonas spp. were influenced by crop rotation, tillage, organic amendments, and chemical seed treatments in subtle but reproducible ways. In no-till corn plots, 2-year rotations with soybean resulted in plants with approximately twofold fewer phlD+ pseudomonads per gram of root, but 3-year rotations with oat and hay led to population increases of the same magnitude. Interestingly, tillage inverted these observed effects of cropping sequence in two consecutive growing seasons, indicating a complex but reproducible interaction between rotation and tillage on the rhizosphere abundance of 2,4-diacetlyphloroglucinol (DAPG) producers. Amending conventionally managed sweet corn plots with dairy manure compost improved plant health and also increased the incidence of root colonization when compared with nonamended plots. Soil pH was negatively correlated to rhizosphere abundance of phlD+ pseudomonads in no-till and nonamended soils, with the exception of the continuous corn treatments. Chemical seed treatments intended to control fungal pathogens and insect pests on corn also led to more abundant populations of phlD in different tilled soils. However, increased root disease severity generally was associated with elevated levels of root colonization by phlD+ pseudomonads in no-till plots. Interestingly, within a cropping sequence treatment, correlations between the relative abundance of phlD and crop stand or yield were generally positive on corn, and the strength of those correlations was greater in plots experiencing more root disease pressure. In contrast, such correlations were generally negative in soybean, a difference that may be partially explained by difference in application of N fertilizers and soil pH. Our findings indicate that farming practices can alter the relative abundance and incidence of phlD+ pseudomonads in the rhizosphere and that practices that reduce root disease severity (i.e., rotation, tillage, and chemical seed treatment) are not universally linked to increased root colonization by DAPG-producers.

Distribution and Fungicide Sensitivity of Fungal Pathogens Causing Anthracnose-like Lesions on Tomatoes Grown in Ohio.

The primary causal agents of anthracnose-like fruit rots in Ohio and their potential resistance to fungicides commonly used to control these fungal pathogens were determined. Nineteen tomato production fields throughout the state were sampled in 2002 and 2003 for fruit with anthracnose-like lesions. Fungi were isolated from these samples, classified using restriction fragment length polymorphism analysis, and identified by internal transcribed spacer sequence analysis. Some of the fungi isolated may represent secondary invaders of preexisting wounds or lesions. Colletotrichum spp. were most abundant in our collection, representing 136 of the 187 isolates. In addition, there were 23 Alternaria, 12 Fusarium, 12 Phomopsis, and 4 Mucor isolates. Colletotrichum, Alternaria, and Fusarium spp. were found throughout the major tomato production areas in the state. In a laboratory investigation, a subset of the Colletotrichum, Alternaria, and Fusarium isolates caused symptoms similar to early development of anthracnose on wounded tomato fruit. In vitro inhibition assays indicated that most Colletotrichum isolates were sensitive to labeled rates of azoxystrobin, chlorothalonil, and mancozeb. However, some Alternaria isolates were less sensitive to azoxystrobin and chlorothalonil than the Colletotrichum isolates. In addition, most Fusarium isolates were also more insensitive to azoxystrobin and mancozeb, and most Phomopsis isolates were not inhibited by azoxystrobin at the levels tested. The patterns of insensitivity to azoxystrobin and chlorothalonil were also observed in situ with excised fruit. Because the fungicides tested are not currently labeled for control of tomato diseases caused by Fusarium or Phomopsis, these results indicate that some pathogen species that can cause anthracnose-like symptoms may not be entirely sensitive to fungicides commonly used in tomato production.

Development of a real-time PCR-based system targeting the 16S rRNA gene sequence for rapid detection of Alicyclobacillus spp. in juice products.

The thermophilic, aciduric Alicyclobacillus spp. are becoming an increasing spoilage concern in the beverage industry. Rapid methods to detect their presence in both raw materials and final products are desirable for industrial quality control. The objective of this study was to develop a real-time TaqMan-based polymerase chain reaction (PCR) system for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., for food industry applications. Two primers and a fluorogenic probe targeting 16S rRNA encoding gene sequences, capable of detecting the genus Alicyclobacillus and a few other closely related thermophiles, were developed, and the efficiency of the detection system was evaluated in both bacterial medium and juice products. Using this system, the presence of less than 100 Alicyclobacillus cells could be detected without cross-reactivity with other common food-borne bacteria.

Minimal changes in rhizobacterial population structure following root colonization by wild type and transgenic biocontrol strains.

Pseudomonas fluorescens strains 2-79, Q8r1-96, and a recombinant strain, Z30-97, produce the antibiotics phenazine-1-carboxylic acid (PCA), 2,4-diacetylphloroglucinol (DAPG), or both antibiotics, respectively. Rhizosphere colonization by these strains and subsequent alterations of bacterial community structure were assayed over multiple growth cycles of wheat under controlled conditions. While added to soil at just log 4 cells per gram prior to planting, all four strains subsequently colonized germinating wheat roots to levels in excess of log 6.5 cells per g (f.w.). Strain-specific differences in rhizosphere competence were observed, but these were not generally related to the chromosomal insertion of the phz genes. Multiple differences in bacterial community structure were detected among treatments in each cycle; however, the large majority of changes were not consistently related to the abundance of inoculant strains in the rhizosphere nor the genetic make-up of the inoculant strains. Nonetheless, T-RFLP profiles of amplified 16S eubacterial sequences indicated that, when compared to the untreated samples, inoculation with Z30-97 resulted in several shifts in rhizosphere bacterial community structure previously associated with decreased levels of root disease.

Microbial populations responsible for specific soil suppressiveness to plant pathogens.

Agricultural soils suppressive to soilborne plant pathogens occur worldwide, and for several of these soils the biological basis of suppressiveness has been described. Two classical types of suppressiveness are known. General suppression owes its activity to the total microbial biomass in soil and is not transferable between soils. Specific suppression owes its activity to the effects of individual or select groups of microorganisms and is transferable. The microbial basis of specific suppression to four diseases, Fusarium wilts, potato scab, apple replant disease, and take-all, is discussed. One of the best-described examples occurs in take-all decline soils. In Washington State, take-all decline results from the buildup of fluorescent Pseudomonas spp. that produce the antifungal metabolite 2,4-diacetylphloroglucinol. Producers of this metabolite may have a broader role in disease-suppressive soils worldwide. By coupling molecular technologies with traditional approaches used in plant pathology and microbiology, it is possible to dissect the microbial composition and complex interactions in suppressive soils.