Analyzing genomic variation in cultivated pumpkins and identification of candidate genes controlling seed traits.

Pumpkins are important vegetable crops widely grown worldwide, and seeds are considered a popular nutraceutical food and an excellent source of protein, oil, and vitamins. Seed size is one of the most important targets for commercial breeding in Cucurbita species; studies have shown that pumpkin seed size variation has a similar trend with fruit size, shape, and seed yield. However, few studies have been conducted to identify genetic loci controlling seed-related traits in cultivated pumpkins. This study analyzed the genomic characteristics of pumpkin breeding materials of 321 Cucurbita accessions collected worldwide, including Cucurbita moschata, Cucurbita maxima, and Cucurbita pepo, using extensive single nucleotide polymorphisms obtained from the genotyping-by-sequencing method, significant genetic variations were identified within and between Cucurbita species. Four major cultivar fruit types were further revealed in C. moschata species, and significant differentiation patterns were detected in several chromosomal regions. A total of 15 significant loci associated with pumpkin seed traits were mapped through a genome-wide association approach; 32 genes previously reported to be associated with seed size regulation in Arabidopsis and Oryza sativa were located in the intervals defined by linkage disequilibrium. Through this study, we gained a deep understanding of the genomic variation distribution across Cucurbita species. The available genetic resources and the associated genetic contents could be used in commercial pumpkin breeding and will facilitate molecular marker-assisted selection in pumpkin seed trait improvement.

Population Dynamics of Xanthomonads Associated with Bacterial Spot of Tomato and Pepper during 27 Years across Taiwan.

Bacterial spot caused by Xanthomonas spp. is the second most important bacterial disease after bacterial wilt of tomato and pepper in Taiwan. To determine the species composition of the Xanthomonas population over 27 years (1989 to 2016) across the country, a large collections of strains from tomato (n = 292) and pepper (n = 198) were examined. In the 1989 to 1999 population, all strains (n = 147) from pepper and 95% strains (n = 198) from tomato were Xanthomonas euvesicatoria. The remaining 5% of strains from tomato were X. vesicatoria. In a 2000 to 2009 population from tomato (n = 36), 22% of the strains were X. perforans and the remaining 78% strains were X. euvesicatoria. In the 2010 to 2016 population, 92% of the strains (n = 50) from pepper were still X. euvesicatoria and the remaining 8% of the strains were X. perforans; however, 99% (n = 58) of the strains from tomato were X. perforans. All of the evaluated (n = 25) strains of X. euvesicatoria collected during 1990 to 2006 were tomato race T1. Four pepper races (P1, P2, P7, and P8) were identified in the X. euvesicatoria population. The strains of X. vesicatoria collected during 1989 to 1999 (n = 8) were tomato race T2 and strains of X. perforans from tomato collected during 2010 to 2016 (n = 12) were race T4 (83%) and race T3 (17%). Four strains of X. perforans from pepper were race T4. All of the strains of X. vesicatoria and X. perforans caused a hypersensitive response in all pepper differentials. Biochemical characterization of representative strains (n = 48) showed that strains of X. euvesicatoria were negative on and amylolytic test and positive on lipase and oxidative-fermentative (OF) tests. The strains of X. vesicatoria were positive on amylolytic and OF tests and were negative on the lipase test. All X. perforans strains showed positive reactions on three tests. Evaluation of the same 48 strains for the sensitivity to copper sulfate (50, 100, 200, 300, and 400 mg liter-1) revealed that the majority of X. euvesicatoria (86%) and X. perforans (94%) strains in the 2010 to 2016 population were tolerant to copper sulfate. The findings suggest that management strategies and breeding programs should consider the new X. perforans species and their new races. The increased number of copper-sulfate-tolerant strains in the 2010 to 2016 population further shows the need for alternative options to copper for managing bacterial spot of tomato and pepper.

Reassessment of QTLs for late blight resistance in the tomato accession L3708 using a restriction site associated DNA (RAD) linkage map and highly aggressive isolates of Phytophthora infestans.

Tomato late blight caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary is a major threat to tomato production in cool and wet environments. Intensified outbreaks of late blight have been observed globally from the 1980s, and are associated with migration of new and more aggressive populations of P. infestans in the field. The objective of this study was to reassess late blight resistance in the wild tomato accession L3708 (Solanum pimpinellifolium L.) against pathogens of different aggressiveness. An F2:3 genetic mapping population was developed using L3708 as the paternal parent. Two isolates of P. infestans, Pi39A and Pi733, were used for inoculation. Pi733 is a highly aggressive genotype that defeats three known late blight resistance genes, Ph-1, Ph-2, and Ph-5t in tomato. In contrast, Pi39A is a less aggressive genotype that defeats only Ph-1. Restriction site Associated DNA Sequencing (RAD-Seq) technology was used to massively sequence 90 bp nucleotides adjacent to both sides of PstI restriction enzyme cutting sites in the genome for all individuals in the genetic mapping population. The RAD-seq data were used to construct a genetic linkage map containing 440 single nucleotide polymorphism markers. Quantitative trait locus (QTL) analysis identified a new disease-resistant QTL specific to Pi733 on chromosome 2. The Ph-3 gene located on chromosome 9 could be detected whichever isolates were used. This study demonstrated the feasibility and efficiency of RAD-Seq technology for conducting a QTL mapping experiment using an F2:3 mapping population, which allowed the identification of a new late blight resistant QTL in tomato.

Improved biovar test for Ralstonia solanacearum.

Race 3, biovar 2 strains of Ralstonia solanacearum are quarantined pathogens in Europe and Canada and Select Agent pathogens in the United States. The biovar classification of R. solanacearum strains is based on their biochemical abilities to utilize a carbohydrate panel. The standard biovar test uses bromothymol blue as a pH indicator in 15 ml culture tubes containing 3 to 5 ml of test media, and takes weeks to complete at 24 or 28 °C. We improved the biovar test by using phenol red as a pH indicator that changes color at a higher pH when a carbohydrate is utilized. We also conducted the test at 32 °C in 0.2 ml of 8-tube strips that reduced the medium needed by at least 20 fold. Using the improved test, biovars of R. solanacearum strains can be determined in 4 days when a panel of seven carbohydrates is used including glucose, trehalose, mannitol, sorbitol, dulcitol, maltose and cellobiose. To differentiate biovars 1, 2, 3 and 4, the test can be further simplified and completed in 3 days using a panel of four carbohydrates containing glucose, trehalose, maltose and dulcitol, significantly saving money, space and time.

Virus-induced gene silencing reveals the involvement of ethylene-, salicylic acid- and mitogen-activated protein kinase-related defense pathways in the resistance of tomato to bacterial wilt.

Bacterial wilt (BW), caused by Ralstonia solanacearum, is a devastating vascular disease of tomato worldwide. However, information on tomato's defense mechanism against infection by this soil-borne bacterium is limited. In this study, virus-induced gene silencing (VIGS) was employed to decipher signaling pathways involved in the resistance of tomato to this pathogen. Defined sequence fragments derived from a group of genes known or predicted to be involved in ethylene (ET) and salicylic acid (SA) signaling transduction pathways and mitogen-activated protein kinase (MAPK) cascades were subjected to VIGS in 'Hawaii 7996', a tomato cultivar with stable resistance to BW, and their effect on resistance was determined. The results indicated that silencing of ACO1/3, EIN2, ERF3, NPR1, TGA2.2, TGA1a, MKK2, MPK1/2 and MPK3 caused significant increase in bacterial proliferation in stembases and/or mid-stems. Partial wilting symptoms appeared on plants in which TGA2.2, TGA2.1a, MKK2 and MPK1/2 were silenced. These results suggested that ET-, SA- and MAPK-related defense signaling pathways are involved in the resistance of tomato to BW. This is the first report elucidating the multiple layers of defense governing the resistance of tomato to BW. The results are discussed to enlighten an important and complex interaction between tomato and a soil-borne vascular pathogen.

Transposon mutagenesis reveals differential pathogenesis of Ralstonia solanacearum on tomato and Arabidopsis.

Ralstonia solanacearum causes a deadly wilting disease on a wide range of crops. To elucidate pathogenesis of this bacterium in different host plants, we set out to identify R. solanacearum genes involved in pathogenesis by screening random transposon insertion mutants of a highly virulent strain, Pss190, on tomato and Arabidopsis thaliana. Mutants exhibiting various decreased virulence levels on these two hosts were identified. Sequence analysis showed that most, but not all, of the identified pathogenesis genes are conserved among distinct R. solanacearum strains. A few of the disrupted loci were not reported previously as being involved in R. solanacearum pathogenesis. Notably, a group of mutants exhibited differential pathogenesis on tomato and Arabidopsis. These results were confirmed by characterizing allelic mutants in one other R. solanacearum strain of the same phylotype. The significantly decreased mutants' colonization in Arabidopsis was found to be correlated with differential pathogenesis on these two plants. Differential requirement of virulence genes suggests adaptation of this bacterium in different host environments. Together, this study reveals commonalities and differences of R. solanacearum pathogenesis on single solanaceous and nonsolanaceous hosts, and provides important new insights into interactions between R. solanacearum and different host plants.

Application of a Preliminary Screen to Select Locally Adapted Resistant Rootstock and Soil Amendment for Integrated Management of Tomato Bacterial Wilt in Taiwan.

Host plant resistance and soil amendment (SA) have not been used extensively to manage tomato bacterial wilt caused by Ralstonia solanacearum due to their variable effects over locations. A preliminary screen was developed to increase the chances of identifying successful control measures under diverse conditions. Isolates from three production areas in Taiwan were collected and their virulence evaluated on tomato. Soil samples from four field sites were collected to evaluate ability to suppress the pathogen of SAs consisting of urea or slaked lime alone or combined at 30°C. The mixture of urea and slaked lime showed the best suppressive effect in three tested soils and was used in subsequent field experiments. Resistant eggplant (EG203) and tomato (Hawaii 7996) rootstocks, selected based on stable resistance against representative strains at the seedling stage, significantly reduced disease incidence in field experiments. EG203 grafted plants exhibited 0 to 2.8% wilted plants compared with 24.4 to 92.9% wilted nongrafted plants. Integrated use of Hawaii 7996 as the rootstock and SA provided significantly greater control of wilt than use of Hawaii 7996 as rootstock alone in only one of the four locations, whereas SA did not provide significant control effect when EG203 was used as the rootstock.

Characterization of a unique chromosomal copper resistance gene cluster from Xanthomonas campestris pv. vesicatoria.

We characterized the copper resistance genes in strain XvP26 of Xanthomonas campestris pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in Xanthomonas and Pseudomonas strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in Pseudomonas syringae pv. tomato and Escherichia coli, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in E. coli and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in Sinorhizobium meliloti, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant X. campestris pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in X. campestris pv. vesicatoria.

Transgenic tomato plants expressing the Arabidopsis NPR1 gene display enhanced resistance to a spectrum of fungal and bacterial diseases.

Development of effective disease-resistance to a broad-range of pathogens in crops usually requires tremendous resources and effort when traditional breeding approaches are taken. Genetic engineering of disease-resistance in crops has become popular and valuable in terms of cost and efficacy. Due to long-lasting and broad-spectrum of effectiveness against pathogens, employment of systemic acquired resistance (SAR) for the genetic engineering of crop disease-resistance is of particular interest. In this report, we explored the potential of using SAR-related genes for the genetic engineering of enhanced resistance to multiple diseases in tomato. The Arabidopsis NPR1 (nonexpresser of PR genes) gene was introduced into a tomato cultivar, which possesses heat-tolerance and resistance to tomato mosaic virus (ToMV). The transgenic lines expressing NPR1 were normal as regards overall morphology and horticultural traits for at least four generations. Disease screens against eight important tropical diseases revealed that, in addition to the innate ToMV-resistance, the tested transgenic lines conferred significant level of enhanced resistance to bacterial wilt (BW) and Fusarium wilt (FW), and moderate degree of enhanced resistance to gray leaf spot (GLS) and bacterial spot (BS). Transgenic lines that accumulated higher levels of NPR1 proteins exhibited higher levels and a broader spectrum of enhanced resistance to the diseases, and enhanced disease-resistance was stably inherited. The spectrum and degree of these NPR1-transgenic lines are more significant compared to that of transgenic tomatoes reported to date. These transgenic lines may be further explored as future tomato stocks, aiming at building up resistance to a broader spectrum of diseases.

Diallel Analysis of Bacterial Wilt Resistance in Tomato Derived from Different Sources.

Bacterial wilt, caused by Ralstonia solanacearum, is a major constraint to tomato production in the tropics and subtropics. Most bacterial wilt-resistant tomato cultivars have not shown consistently high resistance levels over locations. The objective of this study was to determine whether combining resistance derived from different sources would result in F1 progenies with resistance greater than that of the parents. Five bacterial wilt-resistant tomato lines or accessions (CL5915, L285, CRA84, H7997, and GA219), each derived from different resistance sources, and a susceptible processing tomato line (UC204A) were crossed in all combinations without reciprocals. Parents, F1 progenies, and F2 progenies were evaluated in greenhouses at three locations (Taiwan, Philippines, and Indonesia) for percent survival 6 weeks after drench inoculation with virulent local strains of R. solanacearum. Percent survival means over locations were 17.4 to 83.0 for parents and F1 progeny and 16.2 to 75.0 for parents and F2 progeny. The percent survival means over locations of L285 × H7997 were highest among crosses in the F1 (83.0) and F2 (75.0) generations but were not significantly greater than that of H7997. Highly significant mean squares were found in the F1 and F2 progenies for general combining ability (GCA) and GCA × locations. Positive GCA effects over locations were detected for H7997, CRA84, and L285, indicating that progeny with those lines as parents showed bacterial wilt resistance that was greater than the average of all crosses. Only H7997, however, had positive GCA effects estimates at each location for each generation, and its GCA effects estimates over locations were significantly greater than those of the other parents in the F1 and F2 progenies. Among this set of parents, H7997 is the best source to develop bacterial wilt-resistant progeny. We did not observe statistically significant increases in resistance by combining different resistance sources. However, the presence of large GCA variances suggests that hybridization of parents that have high GCA for bacterial wilt resistance, such as H7997, CRA84, or L285, followed by selection in segregating populations might yield inbred progeny with resistance greater than that of the parents.