Seed-borne Bacterial Diseases Research Articles

A CRISPR/LbCas12a-based method for detection of bacterial fruit blotch pathogens in watermelon.

Acidovorax citrulli is the main pathogen causing bacterial fruit blotch, which seriously threatens the global watermelon industry. At present, rapid, sensitive, and low-cost detection methods are urgently needed. The established CRISPR/LbCas12a visual detection method can specifically detect A. citrulli and does not cross-react with other pathogenic bacteria such as Erwinia tracheiphila, Pseudomonas syringae, and Xanthomonas campestris. The sensitivity of this method for genomic DNA detection is as low as 0.7 copies/μL, which is higher than conventional PCR and real-time PCR. In addition, this method only takes 2.5 h from DNA extraction to quantitative detection and does not require complex operation and sample treatment. Additionally, the technique was applied to test real watermelon seed samples for A. citrulli, and the results were contrasted with those of real-time fluorescence quantitative PCR and conventional PCR. The high sensitivity and specificity have broad application prospects in the rapid detection of bacterial fruit blotch bacterial pathogens of watermelon.IMPORTANCEBacterial fruit blotch, Acidovorax citrulli, is an important seed-borne bacterial disease of watermelon, melon, and other cucurbits. The lack of rapid, sensitive, and reliable pathogen detection methods has hampered research on fruit spot disease prevention and control. Here, we demonstrate the CRISPR/Cas12a system to analyze aspects of the specificity and sensitivity of A. citrulli and to test actual watermelon seed samples. The results showed that the CRISPR/Cas12a-based free-amplification method for detecting bacterial fruit blotch pathogens of watermelons was specific for A. citrulli target genes and 100-fold more sensitive than conventional PCR with quantitative real-time PCR. This method provides a new technical tool for the detection of A. citrulli.

Advances in Multi-Omics Approaches for Molecular Breeding of Black Rot Resistance in Brassica oleracea L.

Brassica oleracea is one of the most important species of the Brassicaceae family encompassing several economically important vegetables produced and consumed worldwide. But its sustainability is challenged by a range of pathogens, among which black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is the most serious and destructive seed borne bacterial disease, causing huge yield losses. Host-plant resistance could act as the most effective and efficient solution to curb black rot disease for sustainable production of B. oleracea. Recently, ‘omics’ technologies have emerged as promising tools to understand the host-pathogen interactions, thereby gaining a deeper insight into the resistance mechanisms. In this review, we have summarized the recent achievements made in the emerging omics technologies to tackle the black rot challenge in B. oleracea. With an integrated approach of the omics technologies such as genomics, proteomics, transcriptomics, and metabolomics, it would allow better understanding of the complex molecular mechanisms underlying black rot resistance. Due to the availability of sequencing data, genomics and transcriptomics have progressed as expected for black rot resistance, however, other omics approaches like proteomics and metabolomics are lagging behind, necessitating a holistic and targeted approach to address the complex questions of Xcc-Brassica interactions. Genomic studies revealed that the black rot resistance is a complex trait and is mostly controlled by quantitative trait locus (QTL) with minor effects. Transcriptomic analysis divulged the genes related to photosynthesis, glucosinolate biosynthesis and catabolism, phenylpropanoid biosynthesis pathway, ROS scavenging, calcium signalling, hormonal synthesis and signalling pathway are being differentially expressed upon Xcc infection. Comparative proteomic analysis in relation to susceptible and/or resistance interactions with Xcc identified the involvement of proteins related to photosynthesis, protein biosynthesis, processing and degradation, energy metabolism, innate immunity, redox homeostasis, and defence response and signalling pathways in Xcc–Brassica interaction. Specifically, most of the studies focused on the regulation of the photosynthesis-related proteins as a resistance response in both early and later stages of infection. Metabolomic studies suggested that glucosinolates (GSLs), especially aliphatic and indolic GSLs, its subsequent hydrolysis products, and defensive metabolites synthesized by jasmonic acid (JA)-mediated phenylpropanoid biosynthesis pathway are involved in disease resistance mechanisms against Xcc in Brassica species. Multi-omics analysis showed that JA signalling pathway is regulating resistance against hemibiotrophic pathogen like Xcc. So, the bonhomie between omics technologies and plant breeding is going to trigger major breakthroughs in the field of crop improvement by developing superior cultivars with broad-spectrum resistance. If multi-omics tools are implemented at the right scale, we may be able to achieve the maximum benefits from the minimum. In this review, we have also discussed the challenges, future prospects, and the way forward in the application of omics technologies to accelerate the breeding of B. oleracea for disease resistance. A deeper insight about the current knowledge on omics can offer promising results in the breeding of high-quality disease-resistant crops.

Isolation and Characterization of Bacteriophages Infecting Burkholderia glumae, the Major Causal Agent of Bacterial Panicle Blight in Rice.

Bacterial panicle blight (BPB), caused by Burkholderia glumae, is one of the most severe seed-borne bacterial diseases of rice in the world, which can decrease rice production by ≤75%. Nevertheless, there are few effective measures to manage this disease. In an attempt to develop an alternative management tool for BPB, we isolated and characterized phages from soil and water that are effective to lyse several strains of B. glumae. After tests of host ranges, the phages NBP1-1, NBP4-7, and NBP4-8 were selected for further comprehensive characterization, all of which could lyse B. glumae BGLa14-8 (phage sensitive) but not B. glumae 336gr-1 (phage insensitive). This result indicates that the phages killing B. glumae cells have specific host ranges at the strain level within the bacterial species. In the greenhouse condition of this study, foliar application of the phage NBP4-7 reduced the severity of BPB caused by B. glumae BGLa14-8 ≤62% but did not cause any significant effect on the infection by B. glumae 336gr-1. Electron microscopy and whole-genome sequencing were also performed to characterize the three selected phages. Transmission electron microscopy revealed that the selected phages belong to the family Myoviridae. Furthermore, whole-genome sequence analysis indicated that the three phages belong to a same species and are closely related to the Burkholderia phage KL3, a member of the Myoviridae family.

Bacterial seed endophyte shapes disease resistance in rice.

Cereal crop production is severely affected by seed-borne bacterial diseases across the world. Locally occurring disease resistance in various crops remains elusive. Here, we have observed that rice plants of the same cultivar can be differentiated into disease-resistant and susceptible phenotypes under the same pathogen pressure. Following the identification of a seed-endophytic bacterium as the resistance-conferring agent, integration of high-throughput data, gene mutagenesis and molecular interaction assays facilitated the discovery of the underlying mode of action. Sphingomonas melonis that is accumulated and transmitted across generations in disease-resistant rice seeds confers resistance to disease-susceptible phenotypes by producing anthranilic acid. Without affecting cell growth, anthranilic acid interferes with the sigma factor RpoS of the seed-borne pathogen Burkholderia plantarii, probably leading to impairment of upstream cascades that are required for virulence factor biosynthesis. The overall findings highlight the hidden role of seed endophytes in the phytopathology paradigm of 'disease triangles', which encompass the plant, pathogens and environmental conditions. These insights are potentially exploitable for modern crop cultivation threatened by globally widespread bacterial diseases.

PATHOLOGICAL, BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF THE SEED-BORNE BACTERIA "PANTOEA SPP., XANTHOMONAS SPP. AND PSEUDOMONAS SPP." FROM SOLANACEOUS PLANTS IN EGYPT

The seed-borne bacterial diseases of family solanaceae cause significant economic losses worldwide. In the present study, fourteen bacterial isolates were recovered from seeds of different varieties of tomato, eggplant, black nightshade and tobacco. The seed samples were collected from different locations in Egypt. Isolated bacteria were identified based on morphological, physiological and biochemical tests as well as colonial morphology aspects on the differential medium, yeast extract dextrose calcium carbonate (YDC) besides the selective media (PA 20, King's B and Tween B). Pathogenicity of the isolated bacteria was assessed for causing graywall, bacterial speck and bacterial spot symptoms on tomato seedlings. Disease symptoms were recognized on leaves and stems of the inoculated seedlings. Re-isolation from collected seeds of the inoculated tomato plants was performed. Results indicated that Pantoea spp. was an endophytic bacteria, Pseudomonas spp. was an epiphytic bacteria, while Xanthomonas spp. was both epiphytic and endophytic bacteria. Furthermore, cellular fatty acids composition were identified and quantified to give a profile for tested bacterial isolates. Moreover, the bacterial isolates were identified at the molecular level via PCR reactions utilizing the 16S rRNA gene. Partial DNA sequences were analyzed using BLAST tool revealed that the inferred 16S rRNA partial sequences of the 7 isolates showed similarity to Pantoea ananatis (3 isolates), Pseudomonas syringae pv. tomato (2 isolates) and Xanthomonas vesicatoria (2 isolates). To the best of our knowledge the bacterium associated with the graywall seems to be first report of P. ananatis isolated from tomato fruits and black nightshade seeds in Egypt.

Superior antibacterial activity against seed-borne plant bacterial disease agents and enhanced physical properties of novel green synthesized nanostructured ZnO using Thymbra spicata plant extract

An easy and eco-friendly approach using Thymbra spicata var. spicata L. (TS) plant extract was developed for the formation of nanostructured ZnO. TS aqueous leaf extract was used for the green synthesis of nanostructured ZnO via the Successive ionic layer adsorption and reaction (SILAR) method. Electron microscope images exhibit the morphological adjustments of the samples with respect to change in TS concentration in the growth solution. The nanostructured ZnO grown by SILAR was observed to be polycrystalline with hexagonal crystal structure. The optical energy bandgap value of the samples varies from 3.21 to 3.09 eV as the content of TS increases from 2.5 to 5.0%. Also, the effect of TS additive to ZnO on electrical properties was investigated. It was determined by Van der Pauw measurements that TS contribution to ZnO significantly increased electrical resistance. In addition, impedance analyzes of the produced films were carried out in the frequency range of 20Hz −1 MHz. Nyquist plots showed the single semicircle for all samples, and the values of capacitance and resistance were calculated. Its antibacterial activities was investigated against economically important Gram-positive (Clavibacter michiganensis subsp. Michiganensis) and negative (Pseudomonas syringae pv. Phaseolicola, Pseudomonas cichorii and Pectobacterium carotovorum subsp. Carotovorum) seed-borne plant bacterial disease agents by using paper disc diffusion assay for the first time. In vitro laboratory screenings of green synthesized nanostructured ZnO have given encouraging results, indicating their potential use in the management of seed-borne bacterial diseases.

Transcription Activator-Like Effectors Diversity in Iranian Strains of Xanthomonas translucens.

Bacterial leaf streak caused by different pathovars of Xanthomonas translucens is the most important seedborne bacterial disease of small grain cereals. However, variations in the virulence-associated genomic areas of the pathogen remain uninvestigated. In this study, the diversity of transcription activator-like effectors (TALE) was investigated using the Southern blotting of BamHI-digested genomic DNAs in the Iranian strains of X. translucens. All 65 X. translucens strains were assigned into 13 genotypes, where 57 X. translucens pv. undulosa strains were placed in genotypes 1 to 8, and seven X. translucens pv. translucens strains were placed in genotypes 9 to 12. Interestingly, we did not find any TALE genes in the strain XtKm7 (genotype 13), which showed to be pathogenic only on barley. Virulence and aggressiveness of these strains in greenhouse conditions were in agreement with the TALE-based clustering of the strains in the pathovar level, though variations were observed in the aggressiveness of X. translucens pv. undulosa strains. In general, strains containing higher numbers of putative TALE genes were more virulent on wheat and barley than strains containing fewer. This is the first TALE-based genetic diversity analysis on X. translucens strains and provides novel insights into the virulence repertories and genomic characteristics of the pathogen. Further investigations using TALE mutagenesis and complementation analysis are warranted to precisely elucidate the role of each detected X. translucens TALE in bacterial virulence and aggressiveness either on wheat or barley.

Transcriptome analysis of Xanthomonas oryzae pv. oryzicola exposed to H2O2 reveals horizontal gene transfer contributes to its oxidative stress response.

Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of bacterial leaf streak, is one of the most severe seed-borne bacterial diseases of rice. However, the molecular mechanisms underlying Xoc in response to oxidative stress are still unknown. In this study, we performed a time-course RNA-seq analysis on the Xoc in response to H2O2, aiming to reveal its oxidative response network. Overall, our RNA sequence analysis of Xoc revealed a significant global gene expression profile when it was exposed to H2O2. There were 7, 177, and 246 genes that were differentially regulated at the early, middle, and late stages after exposure, respectively. Three genes (xoc_1643, xoc_1946, xoc_3249) showing significantly different expression levels had proven relationships with oxidative stress response and pathogenesis. Moreover, a hypothetical protein (XOC_2868) showed significantly differential expression, and the xoc_2868 mutants clearly displayed a greater H2O2 sensitivity and decreased pathogenicity than those of the wild-type. Gene localization and phylogeny analysis strongly suggests that this gene may have been horizontally transferred from a Burkholderiaceae ancestor. Our study not only provides a first glance of Xoc's global response against oxidative stress, but also reveals the impact of horizontal gene transfer in the evolutionary history of Xoc.

Evaluation of seedborne bacterial pathogens on common bean cultivars grown in central Anatolia region, Turkey

Bacterial diseases of bean cause economically important losses worldwide. The most important method for managing bacterial diseases on bean is the use of pathogen-free seed. In this study, 198 different dry bean seed samples of six different cultivars including Dermason, Cali, Sira, Battal, Bombay and Seker, were collected from 12 provinces of Central Anatolia Region of Turkey. All were tested to investigate the seedlots as primary inoculum sources of the major bacterial diseases. The data revealed that 22,72 %, 13,63 %, 11,11 %, 1,51 % and 0.5 % of seed samples tested were contaminated with five seedborne bacterial pathogens, Pseudomonas savastanoi pv. phaseolicola (Psp), Pseudomonas syringae pv. syringae (Pss), Xanthomonas axonopodis pv. phaseoli (Xap), X. axonopodis pv. phaseoli var. fuscans (Xapf) and Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), respectively. All bacterial strains isolated were identified based on morphological, physiological, biochemical, molecular and pathogenicity tests. The results showed that Psp and Pss were found together on cv. Cali; Psp and Xap on cv. Dermason and cv. Sira; and Pss and Xap on cv. Seker, cv. Dermason, and cv. Cali. Therefore, the results in the present study suggested that evaluation and selection of pathogen-free seeds are very important for preventing the spread of pathogens and effective management of seed borne bacterial diseases prevalent in bean growing regions; in addition to implementation of integrated crop production strategies such as crop rotation, sanitation, seed treatment, tolerant/resistant cultivar selection and proper bactericide application.

Distribution and severity of bacterial brown spot on dry beans in South Africa: An update

Bacterial brown spot (BBS) of common bean (Phaseolus vulgaris) is a seed-borne bacterial disease caused by Pseudomonas syringae pv. syringae (Pss). Dry bean is an important field crop in South Africa and all commercially available South African dry bean cultivars are susceptible to BBS. The aim of this study was to determine the current distribution and severity of BBS in the dry bean production areas of South Africa. We surveyed 31 locations in five provinces. Disease severity was evaluated on selected plots on a 0–9 scale in four different farming systems: commercial and subsistence farms and strip and national cultivar trials. Leaves with typical BBS symptoms were harvested and transferred to the laboratory. Bacteria were isolated following standard procedure using King’s B medium. Identification of pure isolates was done using physiological and biochemical techniques. Incidence and severity values were used to calculate a disease index. BBS was observed in 88% of locations. BBS incidence was recorded in 54% to 100% of all the farming systems surveyed during the 2008/2009 and 2010/2011 seasons combined. BBS was more severe in the strip and national cultivar trials than on subsistence and commercial farms during the 2008/2009 season and more severe on the commercial farm and in national cultivar trials than on subsistence farms and strip trials during the 2010/2011 season. Findings of this study signal the importance of developing BBS-resistant dry bean cultivars for South Africa.

Implications of bacterial contaminated seed lots and endophytic colonization by Pseudomonas fuscovaginae on rice establishment

The economic impact of seedborne bacterial diseases on rice production provides a major motivation for research on seed health. This paper reports on the endophytic growth of a rifampicin‐marked strain of the seedborne rice pathogen Pseudomonas fuscovaginae. The bacterium was found in most tested seeds indicating that, even without visible discolouration, seed transmission is possible. Crushed discoloured seeds contained more bacterial cells than did non‐crushed discoloured seeds. These bacteria were released during seed soaking, contaminating clean seed and lowering seed germination. Cells of a rifampicin‐resistant strain of P. fuscovaginae, which had been inoculated onto rice seeds, were subsequently recovered from different growth stages and from different rice tissues, thereby indicating endophytic colonization. These results have implications for seedling establishment, as symptomless seeds do not assure disease‐free seeds, and the presence of seedborne bacteria results in poor germination and poor seedling establishment. Elimination of seedborne bacteria by soaking in sodium hypochlorite can increase seed germination. This could be used in developing control strategies, and, if practised regularly, reduce entry of seedborne disease‐causing organisms into crops, resulting in lower disease pressure.

Registration of Common Bean Pinto US14HBR6 Resistant to Race 6 of the Halo Blight Pathogen, Pseudomonas syringae pv. phaseolicola

Halo blight, caused by Pseudomonas syringae pv. phaseolicola (Burkholder) (Psp), is a seed-borne bacterial disease of common bean (Phaseolus vulgaris L.) that is found worldwide, particularly in temperate climates, such as in the midwestern United States. Under favorable conditions, halo blight has been reported to cause up to 45% losses on susceptible cultivars. Nine Psp races have been identified on the basis of the interaction with a series of differentials possessing different combinations of resistance (R) genes. To date, high levels of resistance have been identified for all Psp races with the exception of race 6, which is becoming more prevalent (e.g., in the midwestern United States). Here, we report a source of resistance to Psp race 6 in pinto US 14 (PI 549748). Initially, plants derived from the original US 14 seed stock were variable for their resistance reaction. Thus, a pureline with resistance to Psp race 6, hereafter referred to as US14HBR6 (Reg. No. GP-293, PI 666939), was jointly developed at the University of California, Davis and University of Idaho, Kimberly, through repeated individual plant selections and progeny tests performed under severe halo blight disease pressure. Pinto US14HBR6, like US 14, has an indeterminate prostrate growth habit Type III, white flowers, and 100-seed weight of 23 g. US14HBR6 matures in approximately 95 d in southern Idaho. © Crop Science Society of America. All rights reserved.

Effect of alternative strategies for the disinfection of tomato seed infected with bacterial canker ( Clavibacter michiganensis subsp. michiganensis)

Currently there is a lack of effective seed treatments for bacterial pathogens, with Cu-based compounds (the only chemical treatments permitted under organic farming standards) only providing partial control. The aim of this study was to quantify the effect of alternative treatments for the control of bacterial canker ( Clavibacter michiganensis subsp. michiganensis), a major seed-borne bacterial disease in tomato. Treatments assessed were acidified nitrite (a treatment previously shown to control the seed-borne fungal disease Didymella lycopersici), antagonistic strains of Bacillus spp. and compost extracts, which were not previously evaluated as treatments for seed-borne diseases. Efficacy of treatments was determined in a seed disinfection assay. Ten-minute immersion of seed in 300 mmol l −1 acidified nitrite resulted in 98% being pathogen free. Copper hydroxide, certain strains of Bacillus spp. and all compost extracts resulted in 100% pathogen free seed.

Determination of responses of different bean cultivars against races of Pseudomonas syringae pv phaseolicola, causal agent of halo blight of bean

Use of resistant plant varieties combined with other disease management practices is regarded as the most practical approach to control of seed-borne bacterial disease agents. In this study, responses of different bean cultivars to nine different races of Pseudomonas syringae pv phaseolicola, the causal agent of bacterial halo blight of common bean (Phaseolus vulgaris L.), were determined. During compatible interaction in susceptible cultivars, virulent bacterial races caused water soaked lesion at sites of inoculation. Similar lesions developed in moderately resistant cultivars but symptoms were later associated with more tissue browning around the sites of inoculation. In contrast, the resistant response, produced the characteristic hypersensitive reaction (HR), was characterized as a small discrete browning and tissue collapse at site of inoculation. No local cultivars showed complete resistance to all races tested. Bean cultivars Sehirali-90 and Goynuk-98 were found to be resistant or moderately resistant to five different bacterial races. Bean cultivar, Karacasehir-90, on the other hand, was found to be resistant or moderately resistant to six different bacterial races. Analysis of bacterial growth and the accumulation of isoflavonoid bean phytoalexin, phaseollin in planta were carried out for tissues expressing compatible and incompatible interactions to enable a link to be made between reaction phenotypes and restriction of bacterial growth and phytoalexin accumulations. Development of the HR was clearly associated with the restricted multiplication of bacteria during incompatible interactions. A time-course accumulation analysis on pods treated with different races of bacterial agent showed that a strong correlation was observed between the timing and extent of cell death and accumulation of phaseollin, being rapid and extensive in incompatible interactions compared to compatible interaction.

The Epidemiology and Management of Seedborne Bacterial Diseases

Although seed production has been moved to semiarid regions to escape seedborne pathogens, seedborne bacterial diseases continue to be problematic and cause significant economic losses worldwide. Infested seeds are responsible for the re-emergence of diseases of the past, movement of pathogens across international borders, or the introduction of diseases into new areas. Considerable attention has been paid to improving the sensitivity and selectivity of seed health assays by using techniques such as flow cytometry and the polymerase chain reaction. There has also been progress in understanding infection thresholds and how they influence seed sample size determination and ultimately the reliability of seed health testing. Disease development and dissemination of pathogens from contaminated seedlots can be predicted using formulas that take into account inoculum density and environmental pressures. In general, seeds infested with bacterial pathogens are distributed within a Poisson distribution. In a subset of contaminated seeds, bacteria are distributed in non-Gaussian distributions, e.g., a lognormal distribution.

A method for detection of seedborne bacterial diseases in tomato seeds

A method for detection and quantitative estimation of tomato seedborne pathogenic bacteria has been developed. It enables detection in a 7 g tomato seed sample of as few as ten colony-forming units per gram tomato seeds of the following seedborne pathogens of tomato:Pseudomonas syringae pv. tomato,Pseudomonas corrugata, Xanthomonas campestris pv.vesicatoria, andClavibacter michiganense subsp.michiganense. With representative seed samples, the method employs dry grinding, weighing, bacterial extraction and quantitative calculation on selective or semi-selective medium. The efficiency of this method was tested by diluting pathogen-free seed lots with naturally or artificially infested tomato seeds. This procedure enables one to determine the minimal threshold of pathogen which can be detected by this method on media, in comparison with the percentage of diseased seedlings developed from the same seed lots in the growth chamber or in the greenhouse.

イネ内頴褐変病菌のイネ籾への感染経路

The causal bacterium entered lemmata and paleae through stomata, and multiplied in the intercellular space of the parenchyma. Stomata are mainly open on the inner surface of lemmata and paleae, few on outer surface of lemmata, and they are connected each other through the intercellular space of parenchyma. This infection site does not seem specific phenomenon with regard as in case of Erwinia herbicola. The bacterial grain rot caused by Pseudomonas glumae and the bacterial seedling blight caused by P. plantarii followed this infection pattern to the rice grain. It is suggested that the seed-borne bacterial disease of rice generally follow this course of life cycle. Browning occurred only on the inner surface and the parenchyma of the palea affected with E. herbicola, and is suspected to be one of the defensive reaction of the palea against E. herbicola.

‘Great Northern Valley’ Dry Bean1

Abstract Common blight, caused by the bacterium Xanthomonas phaseoli (E. F. Smith) Dowson, is one of the most serious seed-borne bacterial diseases of beans, Phaseolus vulgaris L. Recommended controls are use of certified disease-free seed and rotation. There is no satisfactory chemical control. Great Northern (GN) cultivars ‘Tara’ (2) and ‘Jules’ (3) have high tolerance to X. phaseoli and high yield but combine the disadvantages of late maturity and vigorous vines, the latter creating conditions favorable for white mold. These 2 cultivars were derived by pedigree selection from the cross of the late maturing, common blight tolerant GN Nebraska #1 sel. 27 line with the early maturing susceptible ‘GN 1140’. The reaction to X. phaseoli was inherited quantitatively (4) while maturity was inherited qualitatively (1). Linkage occurred between genes controlling common blight tolerance and late maturity (4).