Bacterial Fruit Blotch Resistance Research Articles

Genotyping by Sequencing (GBS)-Based QTL Mapping for Bacterial Fruit Blotch (BFB) in Watermelon.

Watermelon (Citrullus lanatus), an economically important and nutritionally rich Cucurbitaceous crop grown worldwide, is severely affected by bacterial fruit blotch (BFB). Development of resistant cultivar is the most eco-friendly, cost-effective, and sustainable way to tackle this disease. This requires wider understanding of the genetics of resistance to BFB. In this study, we identified quantitative trait loci (QTLs) associated with BFB resistance in an F2 mapping population developed from BFB-resistant 'PI 189225' (Citrullus amarus) and -susceptible 'SW 26' (C. lanatus) genotypes based on the polymorphic markers identified by genotyping by sequencing (GSB). A linkage map covering a total genetic distance of 3377.1 cM was constructed. Two QTLs for BFB resistance, namely, ClBFB10.1 and ClBFB10.2, both located on chromosome 10 explaining 18.84 and 15.41% of the phenotypic variations, respectively, were identified. Two SNP-based high-resolution melting (HRM) markers WmBFB10.1 and WmBFB10.2 having high positive correlation with resistance vs. susceptible alleles were developed. The efficacy of the markers was validated in another F2 population derived from SW34 × PI 189225. The highest phenotypic variation was found in the locus ClBFB10.2, which also contains three putative candidate genes for resistance to BFB. These findings will accelerate the development of BFB-resistant watermelon varieties via molecular breeding.

New sources of melon accessions with resistance to bacterial fruit blotch at different phenological stages of melon growth and to multiple strains of Acidovorax citrulli

Bacterial fruit blotch, which is caused by Acidovorax citrulli, is one of the most common diseases of melon crops. To date, there is no disease-resistant commercial cultivar, and management strategies have not been efficient. Therefore, the objectives of this study were to identify melon accessions with genetic resistance to bacterial fruit blotch at different phenological stages of plant growth and to multiple strains of the pathogen. The disease severity was estimated using descriptive scales and evaluated by inoculating A. citrulli into seeds (55 accessions), seedlings, and plants before flowering (38 accessions). After assessing the stability of seven accessions to three strains of the bacterium, two accessions were selected to evaluate their resistance at the flowering and fruiting stages and to perform a transmission assay and optical and scanning electron microscopic analyses. Most accessions showed varied responses (resistance vs. susceptibility) at the different phenological stages of melon growth. However, accessions I-136 and A-43 showed high levels of resistance in most assays and in the stability assay. The fruits of these accessions showed no symptoms of bacterial fruit blotch, and seed transmission of A. citrulli showed values of only 0.6% (I-136) and 2.5% (A-43). Microscopy showed few structural and temporal changes in the colonization of A. citrulli in the leaf mesophyll tissues of these accessions, which can be associated with resistance. The results suggest that accessions A-43 and I-136 can act as donors of bacterial fruit blotch resistance genes, which can be introduced into commercial cultivars through genetic improvement programs.

Screening for bacterial fruit blotch resistance in watermelon fruit

AbstractBacterial fruit blotch (BFB) caused by Acidovorax citrulli is a serious threat to the watermelon industry. Currently, there are no commercial watermelon cultivars with resistance to BFB. In this 3‐yr study, we conducted a screening of 1,452 Citrullus spp. cultigens (PIs and cultivars) for resistance to BFB at the immature fruit stage. However, due to high levels of missing data, many cultigens require additional testing to confirm resistance. Fruit BFB symptoms presented initially as raised bumps, then progressed to blotching, cracking, and internal necrosis. Resistant cultigens rarely proceeded beyond raised bumps at the infection site. Fruit resistance is significantly affected by environmental conditions and has low heritability. Among a subset of cultigens selected for high resistance and susceptibility during the first 2 yr of screening and retested using increased replications, PI 494819 [C. lanatus (Thunb.) Matsum. & Nakai., Zambia], PI 596659 (C. amarus Schrad., South Africa), PI 596670 (C. amarus, South Africa), PI 490384 [C. mucosospermus (Fursa) Fursa, Mali], and PI 596656 (C. amarus, South Africa) had average disease ratings of <1; significantly more resistance than the commercial check, Charleston Gray; low plant‐to‐plant variability within each PI; and infrequent incidence of advanced symptoms. These PIs are potential sources of fruit resistance for breeding programs as well as for research focused on understanding the underlying mechanisms and genetics.

Acidovorax citrulli Type III Effector AopP Suppresses Plant Immunity by Targeting the Watermelon Transcription Factor WRKY6.

Acidovorax citrulli (Ac) is the causal agent of bacterial fruit blotch (BFB), and BFB poses a threat to global watermelon production. Despite its economic importance, the molecular mechanisms underlying Ac pathogenicity and virulence are not well understood, particularly with regard to its type III secreted effectors. We identify a new effector, AopP, in Ac and confirm its secretion and translocation. AopP suppresses reactive oxygen species burst and salicylic acid (SA) content and significantly contributes to virulence. Interestingly, AopP interacts with a watermelon transcription factor, ClWRKY6, in vivo and in vitro. ClWRKY6 shows typical nuclear localization, and AopP and ClWRKY6 co-localize in the nucleus. Ac infection, SA, and the pathogen-associated molecular pattern flg22Ac promote ClWRKY6 production, suggesting that ClWRKY6 is involved in plant immunity and SA signaling. Furthermore, ClWRKY6 positively regulates PTI and SA production when expressed in Nicotiana benthamiana. Importantly, AopP reduces ClWRKY6 mRNA and ClWRKY6 protein levels, suggesting that AopP suppresses plant immunity by targeting ClWRKY6. In summary, we identify a novel effector associated with the virulence mechanism of Ac, which interacts with the transcription factor of the natural host, watermelon. The findings of this study provide insights into the mechanisms of watermelon immune responses and may facilitate molecular breeding for bacterial fruit blotch resistance.

Characterization, identification and expression profiling of genome-wide R-genes in melon and their putative roles in bacterial fruit blotch resistance

BackgroundBacterial fruit blotch (BFB), a disease caused by Acidovorax citrulli, results in significant economic losses in melon. The causal QTLs and genes for resistance to this disease have yet to be identified. Resistance (R)-genes play vital roles in resistance to plant diseases. Since the complete genome sequence of melon is available and genome-wide identification of R-genes has been performed for this important crop, comprehensive expression profiling may lead to the identification of putative candidate genes that function in the response to BFB.ResultsWe identified melon accessions that are resistant and susceptible to BFB through repeated bioassays and characterized all 70 R-genes in melon, including their gene structures, chromosomal locations, domain organizations, motif distributions, and syntenic relationships. Several disease resistance-related domains were identified, including NBS, TIR, LRR, CC, RLK, and DUF domains, and the genes were categorized based on the domains of their encoded proteins. In addition, we profiled the expression patterns of the genes in melon accessions with contrasting levels of BFB resistance at 12 h, 1 d, 3 d, and 6 d after inoculation with A. citrulli. Six R-genes exhibited consistent expression patterns (MELO3C023441, MELO3C016529, MELO3C022157, MELO3C022146, MELO3C025518, and MELO3C004303), with higher expression levels in the resistant vs. susceptible accession.ConclusionWe identified six putative candidate R-genes against BFB in melon. Upon functional validation, these genes could be targeted for manipulation via breeding and biotechnological approaches to improve BFB resistance in melon in the future.

Development of Molecular Marker Linked with Bacterial Fruit Blotch Resistance in Melon (Cucumis melo L.).

Bacterial fruit blotch (BFB) causes losses in melon marketable yield. However, until now, there has been no information about the genetic loci responsible for resistance to the disease or their pattern of inheritance. We determined the inheritance pattern of BFB resistance from a segregating population of 491 F2 individuals raised by crossing BFB-resistant (PI 353814) and susceptible (PI 614596) parental accessions. All F1 plants were resistant to Acidovorax citrulli strain KACC18782, and F2 plants segregated with a 3:1 ratio for resistant and susceptible phenotypes, respectively, in a seedling bioassay experiment, indicating that BFB resistance is controlled by a monogenic dominant gene. In an investigation of 57 putative disease-resistance related genes across the melon genome, only the MELO3C022157 gene (encoding TIR-NBS-LRR domain), showing polymorphism between resistant and susceptible parents, revealed as a good candidate for further investigation. Cloning, sequencing and quantitative RT-PCR expression of the polymorphic gene MELO3C022157 located on chromosome 9 revealed multiple insertion/deletions (InDels) and single nucleotide polymorphisms (SNPs), of which the SNP A2035T in the second exon of the gene caused loss of the LRR domain and truncated protein in the susceptible accession. The InDel marker MB157-2, based on the large (504 bp) insertion in the first intron of the susceptible accession, was able to distinguish resistant and susceptible accessions among 491 F2 and 22 landraces/inbred accessions with 98.17% and 100% detection accuracy, respectively. This novel PCR-based, co-dominant InDel marker represents a practical tool for marker-assisted breeding aimed at developing BFB-resistant melon accessions.

Molecular characterization of Acidovorax citrulli strain NIHHS15-280 causing bacterial fruit blotch disease in Korea and screening of resistance sources in melon

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, is an economically devastating disease in melon. Molecular characterization of causal strains and identification of sources of resistance are necessary to control this important disease. Many strains of BFB were identified by partial sequencing and documented in GenBank. Recently, there was an outbreak of BFB strain NIHHS15-280 in melon fields (Goksung, Jeonnam, Korea) that severely hampered melon production in Korea. For this reason, we characterized NIHHS15-280 by partially sequencing the 16S rRNA gene using PCR-based markers. The partial sequence of the 16S rRNA gene of NIHHS15-280 was much more similar to A. citrulli strains associated with melon and watermelon hosts than other cucurbit hosts. A PCR-based assay identified the strain (NIHHS15-280) as belonging to A. citrulli group I, which predominantly infects non-watermelon cucurbits (virulent to melon). Thirty-five melon genotypes were screened against NIHHS15-280 in a detached-leaf inoculation assay. The lack of visible disease symptoms 11 days after inoculation in lines PI353814 and PI140471 indicated these to be highly resistant lines, whereas moderate resistance was observed in lines PI420145 and SCNU1154 and cultivars ME6 and ME5. Also, the most resistant lines were corroborated by a fruit inoculation assay. The characterized A. citrulli strain NIHHS15-280 (group I) and identified resistant lines will serve as a valuable resource for further genetic studies and improvement of BFB resistance in melon.

QTL mapping of resistance to bacterial fruit blotch in Citrullus amarus.

Six QTLs were associated with affected leaf area in response to inoculation with Acidovorax citrulli in a recombinant inbred line population of Citrullus amarus. Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of cucurbits, has the potential to devastate production of watermelon and other cucurbits. Despite decades of research on host-plant resistance to A. citrulli, no germplasm has been found with immunity and only a few sources with various levels of BFB resistance have been identified, but the genetic basis of resistance in these watermelon sources are not known. Most sources of resistance are plant introductions of Citrullus amarus (citron melon), a closely related species that crosses readily with cultivated watermelon (Citrullus lanatus L.). In this study, we evaluated a recombinant inbred line population (N = 200), derived from a cross between BFB-resistant (USVL246-FR2) and BFB-susceptible (USVL114) C. amarus lines, for foliar resistance to A. citrulli in three replicated greenhouse trials. We found the genetics of BFB resistance to be complicated by strong environmental influence, low heritability and significant genotype-by-environment interactions. QTL mapping of affected leaf area identified six QTL that each explained between 5 and 15% of the variation in BFB resistance in the population. This study represents the first identification of QTL associated with resistance to A. citrulli in any cucurbit.

Effects of nitrogen nutrition on disease development caused by Acidovorax citrulli on melon foliage

Bacterial fruit blotch (BFB) of cucurbits, caused by the seed-borne bacterium Acidovorax citrulli, is a destructive disease that threatens the melon and watermelon industries worldwide. The available means to manage the disease are very limited and there are no reliable sources of BFB resistance. Mineral nutrition has marked effects on plant diseases. To the best of our knowledge, no studies reporting effects of mineral nutrition on BFB severity have been reported to date. In the present study we assessed the influence of nitrogen nutrition on BFB severity and A. citrulli establishment in the foliage of melon plants under greenhouse conditions. Our results show that nitrogen fertilization, based on nitrate only, led to reduced disease severity and bacterial numbers in melon leaves, as compared with two combinations of nitrate and ammonium. No consistent effect of nitrogen nutrition on expression of several plant defense-associated transcripts was found, except for hydroperoxide lyase (HPL), which upon inoculation was repressed to a greater extent under the “nitrate-only” nitrogen regime compared with combined nitrate and ammonium. Reducing BFB severity and A. citrulli establishment in the plant foliage are of particular importance since establishment of the pathogen during the growing season is assumed to increase the incidence of fruit infection, leading to serious yield losses. Further research is needed to elucidate the mechanisms by which nitrogen nutrition influences BFB development, and to assess the effects of nitrogen as well as other minerals on the disease under field conditions.

Bacterial fruit blotch: A threat to the cucurbit industry

Bacterial fruit blotch (BFB) of cucurbits is caused by the Gram-negative bacterium Acidovorax avenae subsp. citrulli. The disease gained importance in the late 1980s, after devastating outbreaks in watermelon fields in several states in the US. Since then, BFB has spread worldwide, and has been reported in other cucurbits such as melon, pumpkin, squash, and cucumber. A. avenae subsp. citrulli is a seedborne pathogen of highly destructive potential. Under favorable conditions, the bacterium spreads rapidly throughout nurseries and in the field, leading to seedling blight or, at a later stage, fruit rot. Strategies for managing BFB are limited and there are no reliable sources of BFB resistance. The disease therefore represents a serious threat to the cucurbit industry. Despite its economic importance, there is little knowledge on basic aspects of the pathogen's biology or on the molecular basis of BFB pathogenesis. Recently, the genome sequence of one A. avenae subsp. citrulli strain was released, making a...