Responsiveness of Candidate Genes on CoPv01CDRK/PhgPv01CDRK Loci in Common Bean Challenged by Anthracnose and Angular Leaf Spot Pathogens.

Anthracnose (ANT) and angular leaf spot (ALS) are significant diseases in common bean, leading to considerable yield losses under specific environmental conditions. The California Dark Red Kidney (CDRK) bean cultivar is known for its resistance to multiple races of both pathogens. Previous studies have identified the CoPv01CDRK/PhgPv01CDRK resistance loci on chromosome Pv01. Here, we evaluated the expression levels of ten candidate genes near the CoPv01CDRK/PhgPv01CDRK loci and plant defense genes using quantitative real-time PCR in CDRK cultivar inoculated with races 73 of Colletotrichum lindemuthianum and 63-39 of Pseudocercospora griseola. Gene expression analysis revealed that the Phvul.001G246300 gene exhibited the most elevated levels, showing remarkable 7.8-fold and 8.5-fold increases for ANT and ALS, respectively. The Phvul.001G246300 gene encodes an abscisic acid (ABA) receptor with pyrabactin resistance, PYR1-like (PYL) protein, which plays a central role in the crosstalk between ABA and jasmonic acid responses. Interestingly, our results also showed that the other defense genes were initially activated. These findings provide critical insights into the molecular mechanisms underlying plant defense against these diseases and could contribute to the development of more effective disease management strategies in the future.

Effect of temperature and humidity on dynamics and transmission of Pseudomonas amygdali pv. lachrymans aerosols.

Cucumber angular leaf spot (ALS) disease, caused by Pseudomonas amygdali pv. lachrymans (Pal), is an emerging disease with a high incidence that causes severe damage to cucumber worldwide. Bacterial aerosols play a crucial role in the epidemiology of greenhouse ALS disease. However, little is known about the influence of temperature and relative humidity (RH) on the dynamics of Pal in aerosols. A study was conducted to investigate the relationships between the concentration of Pal aerosols and their dependence on temperature and RH in aerosol chambers and greenhouses. The results demonstrated that temperature and RH are both significant factors influencing the release amount, survival time and infectivity of Pal in aerosols, while RH has a greater influence on particle size than temperature across the range of conditions tested. The release amount and survival time of Pal in aerosols under high RH (95%) and low temperature (≤ 25°C) conditions were significantly higher than those under low RH (35%) and high temperature (35°C) conditions. The highest release amount of Pal aerosol (96 CFU/m3) and highest survival rate (98.41%) were found at 18°C and 95% RH, while the highest disease index (DI = 60.9) caused by Pal aerosol was found at 25°C and 95% RH. In addition, Pal aerosols presented a larger diameter (4.7->7.0 µm) under high RH (95% RH) than under dry conditions (≤ 65% RH). These findings will play a crucial role in elucidating the influence of environmental parameters on the dynamics and transmission of Pal in aerosols. Based on our findings, preliminary recommendations for controlling airborne Pal spread involve controlling air temperature and RH, which will contribute to the effective alleviation and control of cucumber ALS disease.

Alterations in Primary Carbon Metabolism in Cucumber Infected with Pseudomonas syringae pv lachrymans: Local and Systemic Responses.

The reconfiguration of the primary metabolism is essential in plant-pathogen interactions. We compared the local metabolic responses of cucumber leaves inoculated with Pseudomonas syringae pv lachrymans (Psl) with those in non-inoculated systemic leaves, by examining the changes in the nicotinamide adenine dinucleotides pools, the concentration of soluble carbohydrates and activities/gene expression of carbohydrate metabolism-related enzymes, the expression of photosynthesis-related genes, and the tricarboxylic acid cycle-linked metabolite contents and enzyme activities. In the infected leaves, Psl induced a metabolic signature with an altered [NAD(P)H]/[NAD(P)+] ratio; decreased glucose and sucrose contents, along with a changed invertase gene expression; and increased glucose turnover and accumulation of raffinose, trehalose, and myo-inositol. The accumulation of oxaloacetic and malic acids, enhanced activities, and gene expression of fumarase and l-malate dehydrogenase, as well as the increased respiration rate in the infected leaves, indicated that Psl induced the tricarboxylic acid cycle. The changes in gene expression of ribulose-l,5-bis-phosphate carboxylase/oxygenase large unit, phosphoenolpyruvate carboxylase and chloroplast glyceraldehyde-3-phosphate dehydrogenase were compatible with a net photosynthesis decline described earlier. Psl triggered metabolic changes common to the infected and non-infected leaves, the dynamics of which differed quantitatively (e.g., malic acid content and metabolism, glucose-6-phosphate accumulation, and glucose-6-phosphate dehydrogenase activity) and those specifically related to the local or systemic response (e.g., changes in the sugar content and turnover). Therefore, metabolic changes in the systemic leaves may be part of the global effects of local infection on the whole-plant metabolism and also represent a specific acclimation response contributing to balancing growth and defense.

Characterization of fungal pathogens and germplasm screening for disease resistance in the main production area of the common bean in Argentina.

The common bean (Phaseolus vulgaris L.) is the most important grain legume in the human diet, mainly in Africa and Latin America. Argentina is one of the five major producers of the common bean in the world, and the main cultivation areas are concentrated in the northwestern provinces of this country. Crop production of the common bean is often affected by biotic factors like some endemic fungal diseases, which exert a major economic impact on the region. The most important fungal diseases affecting the common bean in Argentina are white mold caused by Sclerotinia sclerotiorum, angular leaf spot caused by Pseudocercospora griseola, web blight and root rot caused by Rhizoctonia solani, which can cause production losses of up to 100% in the region. At the present, the most effective strategy for controlling these diseases is the use of genetic resistance. In this sense, population study and characterization of fungal pathogens are essential for developing cultivars with durable resistance. In this review we report diversity studies carried out on these three fungal pathogens affecting the common bean in northwestern Argentina, analyzing more than 200 isolates by means of molecular, morphological and pathogenic approaches. Also, the screening of physiological resistance in several common bean commercial lines and wild native germplasm is reviewed. This review contributes to the development of sustainable management strategies and cultural practices in bean production aimed to minimize yield losses due to fungal diseases in the common bean.

Guttation Fluids Containing Pseudomonas amygdali pv. lachrymans Are a Potential Source of Secondary Infection in Cucumber.

Guttation is a common feature of cucumber leaves under high relative humidity conditions; however, little is known about the role of guttation in the transmission of Pseudomonas amygdali pv. lachrymans, which is the pathogen of cucumber angular leaf spot disease. In this study, experimental evidence for the transmission of P. amygdali pv. lachrymans inside cucumber plants and through guttation was provided, and the results proved that P. amygdali pv. lachrymans can be transmitted from the bottom leaf to the upper leaves inside the plant and excreted from the upper leaves through guttation. After that, the third leaf of cucumber was inoculated with P. amygdali pv. lachrymans bacterial suspension, P. amygdali pv. lachrymans was detected on the fifth leaf, the petiole, and the stem and in guttation drops. Healthy cucumber seedlings were infected by P. amygdali pv. lachrymans in the guttation droplets, indicating that guttation fluids containing P. amygdali pv. lachrymans could become a potential source of secondary infection. The results from this study verified the hypothesis that guttation is a potential route for P. amygdali pv. lachrymans excretion from cucumber plants and may be a source of secondary transmission under high relative humidity.

Transcriptome Profiling of Cucumber (Cucumis sativus L.) Early Response to Pseudomonas syringae pv. lachrymans.

Bacterial angular leaf spot disease (ALS) caused by Pseudomonas syringae pv. lachrymans (Psl) is one of the biological factors limiting cucumber open-field production. The goal of this study was to characterize cytological and transcriptomic response of cucumber to this pathogen. Plants of two inbred lines, B10 (susceptible) and Gy14 (resistant), were grown, and leaves were inoculated with highly virulent Psl strain 814/98 under growth chamber conditions. Microscopic and transcriptional evaluations were performed at three time points: before, 1 and 3 days post inoculation (dpi). Investigated lines showed distinct response to Psl. At 1 dpi bacterial colonies were surrounded by necrotized mesophyll cells. At 3 dpi, in the susceptible B10 line bacteria were in contact with degraded cells, whereas cells next to bacteria in the resistant Gy14 line were plasmolyzed, but apparently still alive and functional. Additionally, the level of H2O2 production was higher in resistant Gy14 plants than in B10 at both examined time points. In RNA sequencing more than 18,800 transcripts were detected in each sample. As many as 1648 and 2755 differentially expressed genes (DEGs) at 1 dpi as well as 2992 and 3141 DEGs at 3 dpi were identified in B10 and Gy14, respectively. DEGs were characterized in terms of functional categories. Resistant line Gy14 showed massive transcriptomic response to Psl at 1 dpi compared to susceptible line B10, while a similar number of DEGs was detected for both lines at 3 dpi. This suggests that dynamic transcriptomic response to the invading pathogen may be related with host resistance. This manuscript provides the first transcriptomic data on cucumber infected with the pathovar lachrymans and helps to elucidate resistance mechanism against ALS disease.

Aerosol transmission of Pseudomonas amygdali pv. lachrymans in greenhouses.

Most previous studies on aerosols have focused on the role of aerosols in the transmission of human and animal pathogens; however, little is known about the role of aerosols in the transmission of plant bacterial disease. In this study, experimental evidence for the emission and transmission of Pseunomonas. amygdali pv. lachrymans (Pal) aerosol was provided, and the results supported that diseased cucumber plants served as the main source of Pal aerosol. Bacterial aerosols released by infected plants played a significant role in the epidemiology of cucumber angular leaf spot (ALS) disease. Aerosol chambers were constructed to study the characteristics of Pal aerosols released by artificially infested cucumber plants. The particle size of Pal aerosol was predominately distributed from 1.1 to 4.7 µm, accounting for 72.16% of the total amount of Pal aerosol. The infection threshold of aerosolized Pal to cause ALS disease was 84-179 CFU/m3. In addition, the transmission dynamics of Pal aerosols from donor cucumber plants to recipient cucumber plants were also confirmed in exposure chambers and greenhouses. The results from the present study verified the hypothesis that aerosol dissemination is a potential route for the epidemiology of plant bacterial disease, and these data will contribute to the development of new strategies for the effective alleviation and control of plant bacterial diseases.

Haplotypes at the Phg-2 Locus Are Determining Pathotype-Specificity of Angular Leaf Spot Resistance in Common Bean.

Angular leaf spot (ALS) is one of the most devastating diseases of common bean (Phaseolus vulgaris L.) and causes serious yield losses worldwide. ALS resistance is reportedly pathotype-specific, but little is known about the efficacy of resistance loci against different pathotypes. Here, we report on ALS resistance evaluations of 316 bean lines under greenhouse and field conditions at multiple sites in Colombia and Uganda. Surprisingly, genome-wide association studies revealed only two of the five previously described resistance loci to be significantly associated with ALS resistance. Phg-2 on chromosome eight was crucial for ALS resistance in all trials, while the resistance locus Phg-4 on chromosome 4 was effective against one particular pathotype. Further dissection of Phg-2 uncovered an unprecedented diversity of functional haplotypes for a resistance locus in common bean. DNA sequence-based clustering identified eleven haplotype groups at Phg-2. One haplotype group conferred broad-spectrum ALS resistance, six showed pathotype-specific effects, and the remaining seven did not exhibit clear resistance patterns. Our research highlights the importance of ALS pathotype-specificity for durable resistance management strategies in common bean. Molecular markers co-segregating with resistance loci and haplotypes will increase breeding efficiency for ALS resistance and allow to react faster to future changes in pathogen pressure and composition.

Genetic mapping of psl locus and quantitative trait loci for angular leaf spot resistance in cucumber (Cucumis sativus L.).

One of the most important cucumber diseases is bacterial angular leaf spot (ALS), whose increased occurrence in open-field production has been observed over the last years. To map ALS resistance genes, a recombinant inbred line (RIL) mapping population was developed from a narrow cross of cucumber line Gy14 carrying psl resistance gene and susceptible B10 line. Parental lines and RILs were tested under growth chamber conditions as well as in the field for angular leaf spot symptoms. Based on simple sequence repeat and DArTseq, genotyping a genetic map was constructed, which contained 717 loci in seven linkage groups, spanning 599.7 cM with 0.84 cM on average between markers. Monogenic inheritance of the lack of chlorotic halo around the lesions, which is typical for ALS resistance and related with the presence of recessive psl resistance gene, was confirmed. The psl locus was mapped on cucumber chromosome 5. Two major quantitative trait loci (QTL) psl5.1 and psl5.2 related to disease severity were found and located next to each other on chromosome 5; moreover, psl5.1 was co-located with psl locus. Identified QTL were validated in the field experiment. Constructed genetic map and markers linked to ALS resistance loci are novel resources that can contribute to cucumber breeding programs.

Risks for infection of strawberry plants with an aerosolized inoculum of Xanthomonas fragariae.

Xanthomonas fragariae is the causative agent of angular leaf spot of strawberry, a quarantine organism in plant propagation material in the European Union. Field experiments were conducted to assess the risks for infection of strawberry plants through dispersal of an aerosolized inoculum. In practice, pathogen aerosols can be formed during mowing of an infected crop or by water splashing on symptomatic plants during overhead irrigation or rain. In our experiments, aerosols were generated by spraying suspensions of X. fragariae with a density of 108 cfu ml-1 or water under pressure vertically up into the air. In strawberry plants (cv Elsanta) placed at 1.3, 5 and 10 m distance downwind from the spray boom, infections were found, as evidenced with a combination of dilution-plating and molecular techniques, but more frequently in plants wetted prior to inoculation than in plants kept dry. A logarithmic decrease in infection incidence was found with the distance to the inoculum source. Symptomatic plants were found up to 5 m distance from the inoculum source. No infected plants were found in plants placed 4 m upwind or treated with water. In glasshouse studies, it was shown that under conditions favorable for disease development, spray-inoculation of strawberry plants with estimated densities of X. fragariae as low as 2000 cfu per plant were able to cause symptoms both in cv Elsanta and cv Sonata. Results indicate that there is a considerable risk on infections of strawberry plants exposed to aerosolized inoculum.

Leaf diseases drive the Janzen-Connell mechanism regardless of light conditions: a 3-year field study.

In forests, negative density/distance-dependent seedling mortality (NDD) caused by natural enemies plays a key role in maintaining species diversity [Janzen-Connell (J-C) model]. However, the relative importance of natural enemies in mediating NDD under heterogeneous light conditions has remained unclear. We examined the relative importance of pathogens (i.e., soil pathogens, leaf diseases) on seedling performance in forest understories (FUs) and gaps (gaps) during a 3-year period (results of first year of our study have been previously reported). For the hardwood, Prunus grayana, we investigated seedling mortality, morbidity agents, growth, and root infection by arbuscular mycorrhizal fungi (AMF) beneath conspecific and heterospecific adults in FUs and gaps. Seedling mortality was higher beneath conspecific than heterospecific adults throughout 3 years at both sites, mainly due to continuous leaf disease (i.e., angular leaf spot), whereas damping-off diseases caused mortality only in the first year. Beneath each adult, seedling mortality was higher in FUs than in gaps until second year, but it did not differ between two habitat types in the third year, because leaf diseases caused severe damage even in gaps. Seedling mass was significantly lower beneath conspecific adults. AMF infection of seedlings was also lower beneath conspecific adults, while it was higher in gaps than in FUs beneath both adults. This study demonstrates that the J-C model in a hardwood tree, P. grayana is mainly driven by high NDD seedling mortality caused by airborne leaf diseases, which continuously attack seedlings in a NDD manner regardless of environmental light conditions.

Evaluation of a loop-mediated isothermal amplification assay based on hrpZ gene for rapid detection and identification of Pseudomonas syringae pv. lachrymans in cucumber leaves.

AIMS: Cucumber angular leaf spot caused by Pseudomonas syringae pv. lachrymans (Psl) is an important and destructive disease worldwide, and no effective technique has been developed for the control of the pathogen. Detection of infection or latent in cucumber plants is critical to evaluate disease progress and strengthening management to avoid a serious epidemic in the fields. In this paper, we developed a rapid and sensitive method for detection of Psl using an isothermal method known as loop-mediated amplification (LAMP). METHODS AND RESULTS: A set of six primers was designed to amplify the gene coding for the hrpZ, and conditions for detection were optimized to complete in 60 min at 67°C, and the amplification were confirmed through gel electrophoresis or visually inspected using calcein stain. The specificity of LAMP primers set was widely validated on Psl and nontarget strains. In sensitivity testing, LAMP allowed detection as low as 104  CFU per ml bacterial cells without DNA extraction. The novel method was also applied for detecting Psl in infected cucumber leaves, and even the early onset of disease can be detected by the assay. CONCLUSIONS: This study confirmed that the novel developed LAMP assay is an easy, rapid and sensitive method for the detection of Psl in infected leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is suitable for direct detection of Psl without strain enrichment and complex DNA extraction from samples in the field, and hence it has the capability to be used for on-site disease diagnosis and field surveys.

Inhibition of Xanthomonas fragariae, Causative Agent of Angular Leaf Spot of Strawberry, through Iron Deprivation.

In commercial production settings, few options exist to prevent or treat angular leaf spot (ALS) of strawberry, a disease of economic importance and caused by the bacterial pathogen Xanthomonas fragariae. In the process of isolating and identifying X. fragariae bacteria from symptomatic plants, we observed growth inhibition of X. fragariae by bacterial isolates from the same leaf macerates. Identified as species of Pseudomonas and Rhizobium, these isolates were confirmed to suppress growth of X. fragariae in agar overlay plates and in microtiter plate cultures, as did our reference strain Pseudomonas putida KT2440. Screening of a transposon mutant library of KT2440 revealed that disruption of the biosynthetic pathway for the siderophore pyoverdine resulted in complete loss of X. fragariae antagonism, suggesting iron competition as a mode of action. Antagonism could be replicated on plate and in culture by addition of purified pyoverdine or by addition of the chelating agents tannic acid and dipyridyl, while supplementing the medium with iron negated the inhibitory effects of pyoverdine, tannic acid and dipyridyl. When co-inoculated with tannic acid onto strawberry plants, X. fragariae's ability to cause foliar symptoms was greatly reduced, suggesting a possible opportunity for iron-based management of ALS. We discuss our findings in the context of 'nutritional immunity,' the idea that plant hosts restrict pathogen access to iron, either directly, or indirectly through their associated microbiota.

Patogenesia do óleo essencial e homeopatias de Eucalyptus citriodora em plantas de feijão (Phaseolus vulgaris) / Pathogenesis of essential oil and homeopathies of Eucalyptus citriodora in bean plants (Phaseolus vulgaris)

A homeopatia baseia-se no princípio da cura pelos semelhantes, e na experimentação no indivíduo sadio, doses mínimas e medicamento único. O uso de tratamentos a base de soluções dinamizadas aplicadas em vegetais aumentou nas ultimas décadas, sendo assim necessária a experimentação patogenésica para aplicação de todos os princípios homeopáticos e consolidação da Matéria Vegetal Homeopática. O ensaio patogenésico com plantas de feijão tratadas diariamente via pulverização e irrigação utilizando óleo essencial de Eucalyptus citriodora em diluições (0,5 e 1%) e dinamizados (12 e 30CH) revelaram sintomas externos semelhantes aos provocados por Pseudocercospora griseola, agente causal da mancha angular em feijoeiro. O estudo patogenésico com E. citriodora levanta a possibilidade deste ser utilizado no controle da mancha angular de acordo com o princípio homeopático da cura pela similitude.

Homeopathy is based on the principle of cure by similarity and on the prescription of minimal doses and single medicine to healthy individuals. In plants, performing treatments on the basis of drug dynamisation has increased in the recent decades. In these treatments, it is necessary to conduct a pathogenetic experimentation in order to apply homeopathic principles and to consolidate homeopathic plant material. Through a pathogenetic test with bean plants treated daily by pulverization and irrigation, using diluted and dynamized essential oil of Eucalyptus citriodora - (dilutions: 0.5 and 1%) (oil dynamisation: 12 and 30CH) -, there appeared external symptoms similar to those symptoms caused by Pseudocercospora griseola, i.e., the causal agent of angular leaf spot of bean. The pathogenetics study with E. citriodora raises the possibility of its application to control angular leaf spot in bean plants in accordance with the homeopathic principle of cure by similarity.

Phenotypic recurrent selection in the common bean (Phaseolus vulgaris L) with carioca-type grains for resistance to the fungi Phaeoisariopsis griseola

The efficiency of recurrent selection was assessed in obtaining common bean (Phaseolus vulgaris L) plant lines resistant to the phytopathogenic fungi Phaeoisariopsis griseola. The base bean population was obtained from the partial diallel between seven lines with carioca-type grains and 10 sources of resistance to P. griseola. The plants most resistant to the pathogen were selected in the F2 (S0) generation of the populations (C-0). The best S0:1 plants that presented carioca-type grains were intercrossed to obtain cycle I (C-I). The same procedure was adopted to obtain cycles C-II to C-V. In each recurrent selection cycle, S0:1 progenies selected were also assessed in experiments carried out in Lavras, Brazil, always using as check the Carioca MG (susceptible to P. griseola) and Pérola (tolerant) cultivars. The response to selection for resistance to the pathogen was estimated from the general mean of the S0:1 progenies from each selective cycle compared to the susceptible check Carioca MG. The estimated gain was 6.4 percent per cycle and the indirect response in grain yield by selection for resistance to the pathogen was 8.9 percent per cycle. The variability detected among the progenies in the last selective cycles enabled the prediction of additional responses to recurrent selection.

Ditylenchus drepanocercus Rediscovered in the Neotropics Causing Angular Leaf Spots on Miconia calvescens.

During searches for pathogens to be used as classical biocontrol agents for Miconia calvescens (velvet tree), a devastating plant invader of Hawaii and French Polynesia, damaging angular leaf spots were found repeatedly. The etiological agent of this disease was identified as the nematode Ditylenchus drepanocercus. This nematode has a distinctive falciform appendage at the apex of the tail on both sexes, which allows easy identification. The nematodes were found in the lacunar parenchyma. Infected tissues have abnormally large cells (7 to 13 times the normal size). The lamina at infected areas is chlorotic, slightly thicker, and becomes necrotic with time. The best method of inoculation for this nematode was spraying plants with a suspension containing individuals of various stages on previously wounded leaves. Incubation period was determined to be 20 days.

Disease Progress, Yield Loss, and Control of Xanthomonas fragariae on Strawberry Plants.

The progress of angular leaf spot, caused by the bacterium Xanthomonas fragariae, was examined in field plots of strawberry in the 1994 and 1995 seasons. Disease severity increased intermittently to maxima of circa 25% in 1994 and 15% in 1995. Angular leaf spot reduced marketable yield 8.6% in 1994 and 7.7% in 1995, despite differences in disease severity and base marketable yields for the two seasons. Minimal spread of the pathogen occurred from field plots with inoculated plants to plots with non-inoculated plants. A mixture of cupric hydroxide plus mancozeb was applied at the label rate (1×) at 7- to 14-day intervals and at a reduced rate (0.1×) at 2- to 4-day intervals. The bactericidal mixture at the 1× rate significantly decreased disease, but this mixture was phytotoxic; both plant size and yield were reduced. The 0.1× rate was nonphytotoxic and it reduced disease severity in both years and increased yield in 1994. Lesions of angular leaf spot were detected on strawberry transplants imported from nurseries in Canada and northern United States in both 1993 and 1994. X. fragariae was isolated from those lesions.