Transcriptome-based analysis of candidate gene markers associated with resistance mechanism to Phytophthora melonis that causes root and crown rot in pumpkin.

Root and crown rot incited by an oomycete, Phytophthora melonis , causes significant yield losses in commercial pumpkin (Cucurbita pepo ) production worldwide. Currently, resistant cultivars and knowledge of molecular mechanism of C. pepo against P. melonis are scarce. Here, we analysed the quantitative gene expression changes of 10 candidate gene markers (bHLH87, ERF014, HSF, MYB, PR-1, WRKY21, CPI, POD, PSK, SGT ) in pumpkin roots and leaves at three time points (h post-inoculation, hpi) following inoculation with P. melonis in two resistant (Ghelyani and Tanbal), and two susceptible (Marmari and Khoreshti) varieties of pumpkin. Gene expression using quantitative real time PCR along a time course revealed the strongest transcriptomic response at 48 and 72hpi in resistant genotypes, 1.1-2.7-fold in roots and leaves, respectively, with a high significant correlation (r =0.857**-0.974**). We also found that CPI , PSK, SGT1 and POD act as a dual regulator that similarly modulate immunity not only against P. melonis , but also against other diseases such as early blight (Alternaria cucumerina) , powdery mildew (Podosphaera xanthii ), downy mildews (Pseudoperonospora cubensis ), and pathogenic plant nematodes (Meloidogyne javanica ). Furthermore, significantly higher activities of the ROS scavenging defence enzymes, catalase (1.6-fold increase) and peroxidase (6-fold increase) were observed in the roots of resistant cultivars at different hpi compared with non-inoculated controls. In addition, the biomass growth parameters including leaf and root length, stem and root diameter, root fresh weight and volume were significantly different among studied genotypes. Cumulatively, the transcriptome data provide novel insights into the response of pumpkins for improving pumpkin breeding to P. melonis .

Corrigendum to: Assessing genetic diversity and population structure of Iranian melons (Cucumis melo) collection using primer pair markers in association with resistance to Fusarium wilt.

We evaluated genetic diversity and population structure of Iranian melons (Cucumis melo L.) using combinations of 35 primer pairs: 15 Simple-Sequence-Repeats (SSR); 10 Inter-Simple-Sequence-Repeats (ISSR); and 10 Sequence-related amplified polymorphism (SRAP) markers in association with resistance to melon Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis (FOM ). Genetic similarity was determined by simple matching coefficient (SSM) and dendrogram by clustering-analysis with unweighted pair groups using arithmetic averages (UPGMA). By combining ISSR-SSR-SRAP markers, a high degree of variation among the melons was detected. The mean polymorphism information content (PIC), marker index (MI), effective-number of alleles (I), expected heterozygosity (H), and Nei's gene diversity parameters were 0.392, 0.979, 1.350, 0.551 and 0.225, respectively. According to MI, PIC, I, H, and Nei indices evaluation, ISSR6, ISSR9, SRAP3, SRAP5, SSR3 and SSR6 had the best performance in genetic diversity of the related melons population. The 35 primers yielded a total of 264 bands, of which 142 showed polymorphism. Clustering of genotypes based on resistance to Fusarium wilt, and comparison with grouping on SSR, SRAP and ISSR marker revealed a significant compliance between disease severity and molecular marker dendrograms. Thus, increasing the number of molecular markers for genetic diversity provides a powerful tool for future agricultural and conservation tasks.

Assessing genetic diversity and population structure of Iranian melons (Cucumis melo) collection using primer pair markers in association with resistance to Fusarium wilt.

We evaluated genetic diversity and population structure of Iranian melons (Cucumis melo L.) using combinations of 35 primer pairs: 15 Simple-Sequence-Repeats (SSR); 10 Inter-Simple-Sequence-Repeats (ISSR); and 10 Sequence-related amplified polymorphism (SRAP) markers in association with resistance to melon Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis (FOM ). Genetic similarity was determined by simple matching coefficient (SSM) and dendrogram by clustering-analysis with unweighted pair groups using arithmetic averages (UPGMA). By combining ISSR-SSR-SRAP markers, a high degree of variation among the melons was detected. The mean polymorphism information content (PIC), marker index (MI), effective-number of alleles (I), expected heterozygosity (H), and Nei's gene diversity parameters were 0.392, 0.979, 1.350, 0.551 and 0.225, respectively. According to MI, PIC, I, H, and Nei indices evaluation, ISSR6, ISSR9, SRAP3, SRAP5, SSR3 and SSR6 had the best performance in genetic diversity of the related melons population. The 35 primers yielded a total of 264 bands, of which 142 showed polymorphism. Clustering of genotypes based on resistance to Fusarium wilt, and comparison with grouping on SSR, SRAP and ISSR marker revealed a significant compliance between disease severity and molecular marker dendrograms. Thus, increasing the number of molecular markers for genetic diversity provides a powerful tool for future agricultural and conservation tasks.

Biochemical defense mechanism associated with host-specific disease resistance pathways against Rhizoctonia solani AG3-PT potatoes canker disease.

MAIN CONCLUSION: Screening for resistance in 40 potato genotypes to Rhizoctonia solani AG-3PT-stem-canker, antioxidant enzymes activity as well as total phenol compounds were documented. Rhizoctonia solani AG-3PT-stem-canker is one of the most devastating diseases that leads to severe economic losses in potatoes, Solanum tuberosum globally. Crop management and eugenic practices, especially the use of resistance can be effective in reducing the disease incidence. However, the information about potato-R. Solani interaction is still limited. This study explored screening for resistance in forty potato genotypes to R. solani, analyzing biomass growth parameters (BGPs), as well as antioxidant enzymes activity of which peroxidase/peroxide-reductases (POXs), superoxide dismutase (SOD), polyphenol oxidase (PPO), catalase (CAT), phenylalanine ammonia-lyase (PAL), ß-1,3-glucanase (GLU) and total phenol compounds (TPCs) were taken into account. In addition, we analyzed up-regulation of two gene markers (PR-1 and Osmotin), using reverse transcription quantitative PCR (RT-qPCR). For which, the resistant 'Savalan', partially resistant 'Agria', partially susceptible 'Sagita' and susceptible 'Pashandi' were selected to explore the trails in their roots and leaves over the time courses of 1, 2 and 3-weeks post inoculation (wpi) following inoculation. Cluster analysis divided potatoes into four distinct groups, based on disease severity scales (0-100%) significance. The BGPs, shoot and root length, fresh and dry weight, and root volume were also significantly higher in infected potatoes compared to non-inoculated controls. Antioxidant enzymes activity also indicated the highest increased levels for POX (fourfold at 3wpi), CAT (1.5-fold at 3wpi), SOD (6.8-fold at 1wpi), and PAL (2.7-fold at 3wpi) in the resistant genotype, 'Savalan', whereas the highest activity was recorded in TPC (twofold at 1 wpi), PPO (threefold at 3wpi), and GLU (2.3-fold at 1wpi) in partially resistant genotypes. Although the defense-related enzymatic activities were sharply elevated in the resistant and partially resistant genotypes following inoculation, no significant correlations were between the activity trends of the related enzymes. The two related gene markers also showed comprehensive transcriptional responses up to 3.4-fold, predominantly in resistant genotypes. Surprisingly, the PR-1 gene marker, basically resistant to Wilting agent Verticillium dahlia was overexpressed in resistant 'Savalan' and 'Agria' against R. solani AG3-PT. Similar results were obtained on Osmotin gene marker resistant to late-blight P. infestans, and early-blight Alternaria solani that similarly modulates immunity against R. solani. Furthermore, there was a significant correlation between resistance, enzyme activity, and gene expression in the aforesaid cultivars. Studying the physiological metabolic pathways of antioxidant enzymes activity appears to be an important direction in research to elucidate resistance to R. solani in potatoes.

Identification of novel associations of candidate genes with resistance to Rhizoctonia solani AG-3PT in Solanum tuberosum stem canker.

To develop an understanding mechanism to define responding of potatoes to R. solani, we analyzed the expression of ten novel candidate gene-markers using reverse-transcription-quantitative PCR (RT-qPCR) in resistant 'Savalan' and partially resistant 'Agria' in contrast to susceptible 'Sagita', and partially susceptible 'Pashandi'. In addition, oxidant-enzymatic-activity of catalase and superoxide-dismutase, as well as biomass-growth-parameters; shoot and root length, fresh and dry weight, and root volume were considered as complementary factors to the involving mechanism accordingly. Gene-markers up-regulated maximum up to 3.5-fold with the highest correlation, r = 0.939** following R. solani-inoculation, predominantly in resistant genotypes. Surprisingly, WRKY8-gene, basically resistant to late-blight-Phytophtora infestans was also up-regulated to 2.3-fold in resistant 'Savalan' followed by 'Agria'. Similar results with 3.1-fold were obtained on Osmotin-gene resistant to early-blight-Alternaria alternata. Enzymatic-activity of catalase with 1.6-fold and superoxide-dismutase, 6.8-fold also showed the highest level of activity in resistant genotypes, and had a high significant correlation, r = 773** and r = 0.881** with expression levels of related gene-markers respectively. Similarly, there were significant differences in biomass-growth-parameters, but with reductions in partially susceptible 'Sagita' and susceptible 'Pashandi'. Conclusively, S. tuberosum-R. solani interaction revealed that certain gene-markers can cover resistance to more than one disease simultaneously.

Genomic markers analysis associated with resistance to Alternaria alternata (fr.) keissler-tomato pathotype, Solanum lycopersicum L.

Alternaria alternata, the causal pathogen of early blight (EB) disease, is one of the most important diseases in tomato, and other solanaceae family. We analyzed 35 tomato genotypes for quantitative/qualitative traits and biomass growth parameters, as well as the extent and structure of genetic variation associated with EB resistance. Phenotypic comparisons displayed significant differences in leaf blade width (24.95%), stem thickness (30.28%), foliage density (18.88%), and plant size (18.89%), with significant positive correlations with EB resistance (0.18-0.75). Correlation analysis showed that mature fruit size, thickness of fruit pericarp, and leaf type were significantly and negatively correlated with EB resistance (up to -0.41). The susceptible tomato seedlings represented significant reductions in biomass parameters. According to ISSR analysis, the highest resolving power (≥0.79) and heterozygosity (≥0.24) values revealed the presence of high genetic variability among the tomato genotypes. Bayesian model-based STRUCTURE analysis assembled the genotypes into 4 (best ΔK = 4) genetic groups. Combined phenotypic and molecular markers proved to be significantly useful for genetic diversity assessment associated with EB disease resistance.

Genetic diversity and biochemical analysis of Capsicum annuum (Bell pepper) in response to root and basal rot disease, Phytophthora capsici.

This study analyzed the genetic variability and biochemical characteristics of edible and ornamental accessions of pepper, Capsicum annuum, in response to root and basal rot disease (RCR), caused by Phytophthora capsici, using resistance screening and genetic variability via Inter Simple Sequence Repeat marker (ISSR), bio-mass parameters, and enzymatic activity of Peroxidase or peroxide reductases (POX), Superoxide dismutase (SOD), Polyphenol oxidase (PPOs), Catalase (CAT), Phenylalanine ammonia-lyase (PAL), ß-1,3-glucanase and phenolic content. The resistance in C. annuum '37ChilPPaleo', '19OrnP-PBI' and '23CherryPOrsh' and susceptibility in '2BP-PBI', '24BP-301' and '26BPRStarlet' accessions were confirmed. Nineteen out of 21 ISSR primers generated 185 polymorphic bands with a mean percentage band of 98.5 %, and an average number of bands of 9.9 per primer. Biomass parameters were significantly higher in resistant genotypes than the susceptible ones and non-inoculated controls. All the seven candidate enzymes were highly up-regulated in the resistant C. annuum accessions '19OrnP-PBI', '37ChillP-Paleo' and '23CherryP-Orsh' inoculated with P. capsici The mean level of enzyme activity varied from 1.5 to 5.6-fold higher in the resistant C. annuum, of which SOD was increased by 5.6 fold, followed by PAL 4.40 and PPO 3.75 fold in comparison to susceptible and non-inoculated controls. Overall, there was no significant correlation between resistance and genetic variability, and also between genetic variability and enzyme activity levels. However, there was a highly significant correlation between the resistance, bio-mass parameters and enzyme activity levels.

Expression analysis of defense-related genes in cucumber (Cucumis sativus L.) against Phytophthora melonis.

Phytophthora melonis is one of the most destructive cucumber disease, causing severe economic losses in the globe. Despite intense research efforts made in the past years, no permanent cure currently exists for this disease. With the aim to understand the molecular mechanisms of defense against P. melonis, root collars and leaves of four cucumber genotypes consisting of resistant Ramezz; moderately resistant Baby, and very susceptible Mini 6-23 and Extrem, were monitored for quantitative gene expression analysis of the five antifungal and/or anti-oomycete genes (CsWRKY20, CsLecRK6.1, PR3, PR1-1a and LOX1), at three points after inoculation with P. melonis. The gene expression analysis indicated, P. melonis strongly enhanced the expression of these genes after inoculation, in both the leaves and root collars. Further, not only the transcript levels of these genes were significantly higher in resistant and moderately resistance genotypes, but also the time point of the highest relative expression ratio for the five genes was different in the four cucumber genotypes. CsWRKY20 and PR3 showed the maximum expression in Ramezz at 48 h post inoculation (hpi) while CsLecRK6.1, and LOX1 showed the highest expression at 72 hpi. In addition, PR1-1a showed the maximum expression in the Baby at 72 hpi. Root collars responded faster than leaves, and some responses were more strongly up-regulated in root collars than in leaves. The genes found to be involved in disease resistance in two different organs of cucumber after pathogen infection. The results suggest that increased expression of these genes led to activation of defense pathways, and could be responsible for a reduced P. melonis colonization capacity in Ramezz and Baby. Overall, this work represents a valuable resource for future functional genomics studies to unravel molecular mechanisms of Cucumis sativus-P. melonis interaction.

Regulation of related genes promoting resistant in Iris against root rot disease, Fusarium oxysporum f. sp. gladioli.

Iris is one of the most popular and best-selling ornamental plants around the globe. Fusarium root rot disease, Fusarium oxysporum f.sp. gladioli (FOG) is one of the most serious disease of Iridaceae and Iris plants. In this study, three resistant and three susceptible Iris genotypes were inoculated with FOG isolates to evaluate expression of related genes promoting defense to disease at intervals times at two, four and six weeks post inoculation. Total RNA was extracted using an AccuZol™ reagent, and the first-strand Cdna was synthesized accordingly. Expression level of WRKY transcription factors (WRKY), lectin receptor kinase (LecRK), pathogenesis-related protein (PR3), lipoxygenase (LOX1) and ribosome-inactivating proteins (RIP) genes was investigated using quantitative polymerase chain reaction (qPCR). The transcriptional level of five defense-related genes were up-regulated in FOG-infected samples. The genes expression in resistant Iris genotypes NIOP3, NIOP15 and NIOP16 was much higher than susceptible NIOP1, NIOP12 and NIOP20 genotypes. The highest level of expression was observed in all the genes and genotypes at 6 weeks post inoculation. The phenotypic symptoms of genotypes and changes in the expression of genes confirmed resistance in Iris genotypes NIOP3, NIOP15 and NIOP16 in comparison to susceptible genotypes NIOP1, NIOP12 and NIOP20, and un-inoculated control Iris plants. Identifying disease-resistant genotypes can contribute to the development of new ornamental cultivars that can be deployed to ensure high quality and lasting Iris plants.

Analysis of candidate genes expression associated with defense responses to root and collar rot disease caused by Phytophthora capsici in peppers Capsicum annuum.

Root and collar rot disease caused by Phytophthora capsici (Leonian) is one of the most serious diseases in pepper, Capsicum annuum L. Knowledge about resistant genes is limited in pepper accessions to P. capsici. In this study, a diverse collection of 37 commercial edible and ornamental genotypes, and implication of seven novel candidate DEGs genes (XLOC_ 021757, XLOC_021821, XLOC_012788, XLOC_011295, XLOC_021928, XLOC_015473 and XLOC_000341) were up-regulated on resistant and susceptible pepper cultivars, through real-time polymerase chain reaction (qPCR) at transplanting and maturing stages. All seven related defense-gene candidates were up-regulated in all inoculated accessions to P. capsici, but these genes were highly expressed in resistant ones, 19OrnP-PBI, 37ChillP-Paleo, and 23CherryP-Orsh. The transcriptional levels of the seven related candidate DEGs were 5.90, 5.64, 5.62, 5.18, 3.94, 3.69, 3.16 folds higher in the resistant pepper genotypes, than the control ones, non-inoculated genotypes respectively. The candidate genes expressed herein, will provide a basis for further gene cloning and functional verification studies, and also will aid in an understanding of the regulatory mechanism of pepper resistance to P. capsici.

Genetic defense analysis of tomatoes in response to early blight disease, Alternaria alternata.

Early blight disease of tomato is one of the most devastating biotic stresses worldwide, and in Iran, Alternaria alternata is one of the most predominant species causing the disease. In the current study, a diverse collection of 35 tomato genotypes and implication of 5 SlWRKYs and 7 PR genes as well as enzymatic activity were evaluated on resistant and susceptible cultivars through real-time polymerase chain reaction at transplanting and maturing stages and by measuring product formation using spectrophotometry. The results indicated that the expression of these antifungal genes in 14 genotypes at two growth stages after inoculation with A. alternata highly enhanced by 1-50-fold. There was also significant upregulation of WRKYs and PRs genes among the resistant tomato varieties in comparison to susceptible and control varieties at both stages. These findings demonstrate the varieties that showed increased or decreased SlWRKY1 expression also displayed similar changes in the expression of PR1 and PR2 genes. Furthermore, the differential expression patterns of SlWRKY1 and SlWRKY11 were consistent with PR7 and PDF1.2 expression patterns. The analysis of enzymatic activity of PR2 and PR3 proteins, ß-1,3-glucanase, and chitinase showed the highest level of activity in resistant inoculated genotypes against A. alternata. Therefore, the current findings suggest the possible involvement of these transcription factors in the increased expression of PR genes in response to A. alternata infection.

A pilot randomized controlled trial on the effectiveness of inclusion of a distant learning component into empathy training.

BACKGROUND: Studies have shown a gradual decline in empathy of medical trainees with increasing years of education. METHODS to augment empathy show some promise, but the most effective methods are both expensive and time consuming. To assess effectiveness of communication skills training program as a distant learning method in improving empathy. METHODS: Fourteen first year residents of psychiatry were randomly allocated to either participate in a two day workshop on communications skills (attending group) or to watch the videotape of the first day and participate in the second day (distance learning group). Assessments included Jefferson Scale of Empathy (JSE) and objective assessment of empathy (OAE) during a simulated interview, before and 3 months after the training. RESULTS: The empathy was significantly increased in the attending group as measured by OAE. The score of JSE also increased in the attending group but did not reach a significance level. No increase in empathy was seen in the distance learning group. CONCLUSION: Watching the videotape of the workshop is not effective in improving empathy of residents. More interactive methods should be sought if we plan to use distance learning methods in enhancement of empathy.

Quality of life, religious attitude and cancer coping in a sample of Iranian patients with cancer.

BACKGROUND: Cancer is one of the leading causes of mortality and morbidity worldwide. The incidence of cancer has increased markedly in recent decades in most countries. Studies have shown that diseases such as cancer affect the individuals' quality of life. METHODS: The sample of study consisted of 384 patients selected through non-random convenient sampling procedure from three general hospitals and outpatient clinics in Isfahan and Tehran. The measures used in the study included a demographic questionnaire, the Iranian version of the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30), the Cancer Coping Questionnaire, and the Religious Attitude Questionnaire. RESULTS: The results revealed significant correlation between patients' scores on the total scale of the Cancer Coping Questionnaire and their scores on the Global health status/Quality of Life. Significant correlations were also found between patients' scores on the Religious Attitude Questionnaire and various scales of the Quality of Life Questionnaire. However, no significant correlations were found between Cancer Coping and Religious Attitude measures in any type of cancer except for the prostate cancer. CONCLUSIONS: Religious attitude was a significant and important factor in coping with cancer. In addition, patients' quality of life correlated significantly with religious attitude as well as cancer coping measures. However, the results did not show any significant relationship between religious attitude and cancer coping measures except in patients with prostate cancer. The findings of this study are consistent with other studies that have shown significant correlations between religiosity and spirituality and quality of life in patients with life threatening diseases.