Polysaccharides from Dendrobium officinale improve obesity-induced insulin resistance through the gut microbiota and the SOCS3-mediated insulin receptor substrate-1 signaling pathway.

BACKGROUND: Obesity induces insulin resistance and chronic inflammation, impacting human health. The relationship between obesity, gut microbiota, and regulatory mechanisms has been studied extensively. Dendrobium officinale polysaccharide (DOP), a traditional Chinese herbal medicine, potentially reduces insulin resistance. However, the mechanism through which DOP affects gut microbiota and alleviates obesity-induced insulin resistance in rats requires further investigation. RESULTS: The current study aimed to assess the impact of DOP on gut microbiota and insulin resistance in rats on a high-fat diet. The results revealed that DOP effectively reduced blood lipids, glucose disorders, oxidative stress, and inflammatory infiltration in the liver of obese Sprague Dawley rats. This was achieved by downregulating SOCS3 expression and upregulating insulin receptor substrate-1 (IRS-1) by regulating the JAK/STAT/SOCS3 signaling pathway. Notably, DOP intervention enhanced the abundance of beneficial gut microbiota and reduced harmful microbiota. Correlation analysis demonstrated significant associations among intestinal microbiota, SOCS3-mediated IRS-1 expression, and inflammatory factors. CONCLUSION: Dendrobium officinale polysaccharide regulated the gut microbiota, enhanced IRS-1 expression, and mitigated liver injury and insulin resistance due to a high-fat diet. These findings depict the potential anti-insulin resistance properties of DOP and offer further evidence for addressing obesity and its complications. © 2023 Society of Chemical Industry.

Histone H3 gene is not a suitable marker to distinguish Alternaria tenuissima from A. alternata affecting potato.

Potato Alternaria leaf blight is one of the economically most important disease in potato production worldwide. A recent study reported a quick method to distinguish main Alternaria pathogens A. tenuissima, A. alternata, and A. solani using partial histone H3 gene sequences. Using this method, our collection of 79 isolates from 8 provinces in China were presumably separated into A. tenussima and A. alternata. But in depth morphological and genetic analysis casted doubt on this identification. Culture morphologies of six presumed A. alternata isolates (PresA_alt) and six presumed A. tenuissima isolates (PresA_ten) were not significantly different. PresA_ten isolates also produced conidia in branched chains which supposed to be A. aternata. Phylogenetic analyses were conducted using internal transcribed spacer region (ITS) and five genes commonly used for species identification including glyceraldehyde-3-phosphate dehydrogenase (GPDH), translation elongation factor 1-alpha (TEF1), ß-tubulin, plasma membrane ATPase (ATPase), and calmodulin genes. The results showed that GPDH and TEF1 sequences of PresA_alt and PresA_ten isolates were identical. The 12 isolates did not cluster by presumed species neither by individual or concatenated sequence comparisons. The phylogeny-trait association analysis confirmed that the two group isolates were undistinguishable by those molecular markers. Analysis of histone H3 gene sequences revealed variable intron sequences between PresA_ten and PresA_alt isolates, but the amino acid sequences were identical. Our results indicate that the previously published method to distinguish Alternaria species based on histone H3 gene sequence variation is inaccurate and that the prevalence of A. tenuissima isolates in China was likely overestimated.

Induced expression of CYP51 associated with difenoconazole resistance in the pathogenic Alternaria sect. on potato in China.

BACKGROUND: Early blight caused by Alternaria spp. is amongst the most important diseases in potato. Demethylation inhibitor (DMI) fungicides are widely used to control the disease but long-term use may decrease its control efficacy due to fungicide resistance. This study investigated the occurrence of difenoconazole resistance in Alternaria spp. and molecular resistant mechanisms. RESULTS: EC50 values of 160 isolates to difenoconazole ranged from 0.026 µg mL-1 to 15.506 µg mL-1 and the frequency of difenoconazole sensitivity formed a non-normal distribution curve with a major and a minor peak. Isolates with EC50 values of 4.121 and 5.461 µg mL-1 were not controlled effectively at fungicide doses of 50 and 100 µg mL-1 . Cross-resistance was observed between DMI fungicides difenoconazole and propiconazole, but not between difenoconazole and other fungicide groups, including boscalid, iprodione, or carbendazim. The CYP51gene was 1673 bp encoding 525 amino acids in length and contained two introns. All sensitive and resistant isolates had the identical amino acid sequence of CYP51, with the exception of one resistant isolate carrying a mutation of R511W. A 6 bp insertion in the upstream region was observed in half of the resistant isolates. In the absence of propiconazole, the relative expression of CYP51 was not significantly different in sensitive and resistant isolates. In the presence of difenoconazole, expression of CYP51 gene was induced significantly in the DMI-resistant isolates but not in the sensitive ones. CONCLUSION: Induced expression of CYP51 in resistant isolates exposed to difenoconazole is an important determinant for DMI resistance in potato pathogens Alternaria sect. © 2019 Society of Chemical Industry.

Cross-resistance of the pathogenic fungus Alternaria alternata to fungicides with different modes of action.

BACKGROUND: Cross-resistance, a phenomenon that a pathogen resists to one antimicrobial compound also resists to one or several other compounds, is one of major threats to human health and sustainable food production. It usually occurs among antimicrobial compounds sharing the mode of action. In this study, we determined the sensitivity profiles of Alternaria alternata, a fungal pathogen which can cause diseases in many crops to two fungicides (mancozeb and difenoconazole) with different mode of action using a large number of isolates (234) collected from seven potato fields across China. RESULTS: We found that pathogens could also develop cross resistance to fungicides with different modes of action as indicated by a strong positive correlation between mancozeb and difenoconazole tolerances to A. alternata. We also found a positive association between mancozeb tolerance and aggressiveness of A. alternata, suggesting no fitness penalty of developing mancozeb resistance in the pathogen and hypothesize that mechanisms such as antimicrobial compound efflux and detoxification that limit intercellular accumulation of natural/synthetic chemicals in pathogens might account for the cross-resistance and the positive association between pathogen aggressiveness and mancozeb tolerance. CONCLUSIONS: The detection of cross-resistance among different classes of fungicides suggests that the mode of action alone may not be an adequate sole criterion to determine what components to use in the mixture and/or rotation of fungicides in agricultural and medical sects. Similarly, the observation of a positive association between the pathogen's aggressiveness and tolerance to mancozeb suggests that intensive application of site non-specific fungicides might simultaneously lead to reduced fungicide resistance and enhanced ability to cause diseases in pathogen populations, thereby posing a greater threat to agricultural production and human health. In this case, the use of evolutionary principles in closely monitoring populations and the use of appropriate fungicide applications are important for effective use of the fungicides and durable infectious disease management.

A gold nanoparticle-based lateral flow biosensor for sensitive visual detection of the potato late blight pathogen, Phytophthora infestans.

Phytophthora infestans, the causal agent of late blight in potatoes and tomatoes, is the most important and ongoing pathogenic threat to agricultural production worldwide. Rapid and early identification of P. infestans is an essential prerequisite for countering the further spread of infection. In this study, a novel method for visual detection of P. infestans has been developed by integrating universal primer mediated asymmetric PCR with gold nanoparticle (AuNP)-based lateral flow biosensor. We employed asymmetric PCR to generate large amounts of single-stranded DNA (ssDNA) by amplifying a region of P. infestans-specific repetitive DNA sequence. The ssDNA products were then applied to the lateral flow biosensor to perform a visual detection using sandwich-type hybridization assays. In the presence of target DNA, sandwich-type hybridization reactions among the AuNP-probe, target DNA and capture probe were performed on the test line of the biosensor, and then a characteristic red band was produced for the accumulation of AuNPs. Quantitative analysis obtained by recording the optical intensity of the red band demonstrated that this biosensor could detect as little as 0.1 pg µL-1 genomic DNA. Furthermore, the specificity of the biosensor was confirmed by detecting three other Phytophthora species and two pathogenic fungi. We believe this method has potential application in early prediction of potato late blight disease and instigation of management actions to reduce the risk of epidemic development.

RXLR Effector AVR2 Up-Regulates a Brassinosteroid-Responsive bHLH Transcription Factor to Suppress Immunity.

An emerging area in plant research focuses on antagonism between regulatory systems governing growth and immunity. Such cross talk represents a point of vulnerability for pathogens to exploit. AVR2, an RXLR effector secreted by the potato blight pathogen Phytophthora infestans, interacts with potato BSL1, a putative phosphatase implicated in growth-promoting brassinosteroid (BR) hormone signaling. Transgenic potato (Solanum tuberosum) plants expressing the effector exhibit transcriptional and phenotypic hallmarks of overactive BR signaling and show enhanced susceptibility to P. infestans Microarray analysis was used to identify a set of BR-responsive marker genes in potato, all of which are constitutively expressed to BR-induced levels in AVR2 transgenic lines. One of these genes was a bHLH transcription factor, designated StCHL1, homologous to AtCIB1 and AtHBI1, which are known to facilitate antagonism between BR and immune responses. Transient expression of either AVR2 or CHL1 enhanced leaf colonization by P. infestans and compromised immune cell death activated by perception of the elicitin Infestin1 (INF1). Knockdown of CHL1 transcript using Virus-Induced Gene Silencing (VIGS) reduced colonization of P. infestans on Nicotiana benthamiana Moreover, the ability of AVR2 to suppress INF1-triggered cell death was attenuated in NbCHL1-silenced plants, indicating that NbCHL1 was important for this effector activity. Thus, AVR2 exploits cross talk between BR signaling and innate immunity in Solanum species, representing a novel, indirect mode of innate immune suppression by a filamentous pathogen effector.

Increased frequency of self-fertile isolates in Phytophthora infestans may attribute to their higher fitness relative to the A1 isolates.

Knowledge of population dynamics of mating types is important for better understanding pathogen's evolutionary potential and sustainable management of natural and chemical resources such as host resistances and fungicides. In this study, 2250 Phytophthora infestans isolates sampled from 61 fields across China were assayed for spatiotemporal dynamics of mating type frequency. Self-fertile isolates dominated in ~50% of populations and all but one cropping region with an average frequency of 0.64 while no A2 isolates were detected. Analyses of 140 genotypes consisting of 82 self-fertile and 58 A1 isolates indicated that on average self-fertile isolates grew faster, demonstrated higher aggressiveness and were more tolerant to fungicides than A1 isolates; Furthermore, pattern of association between virulence complexity (defined as the number of differential cultivars on which an isolate can induce disease) and frequency was different in the two mating types. In A1 isolates, virulence complexity was negatively correlated (r = -0.515, p = 0.043) with frequency but this correlation was positive (r = 0.532, p = 0.037) in self-fertile isolates. Our results indicate a quick increase of self-fertile isolates possibly attributable to their higher fitness relative to A1 mating type counterpart in the field populations of P. infestans in China.

Trade-offs and evolution of thermal adaptation in the Irish potato famine pathogen Phytophthora infestans.

Temperature is one of the most important environmental parameters with crucial impacts on nearly all biological processes. Due to anthropogenic activity, average air temperatures are expected to increase by a few degrees in coming decades, accompanied by an increased occurrence of extreme temperature events. Such global trends are likely to have various major impacts on human society through their influence on natural ecosystems, food production and biotic interactions, including diseases. In this study, we used a combination of statistical genetics, experimental evolution and common garden experiments to investigate the evolutionary potential for thermal adaptation in the potato late blight pathogen, Phytophthora infestans, and infer its likely response to changing temperatures. We found a trade-off associated with thermal adaptation to heterogeneous environments in P. infestans, with the degree of the trade-off peaking approximately at the pathogen's optimum growth temperature. A genetic trade-off in thermal adaptation was also evidenced by the negative association between a strain's growth rate and its thermal range for growth, and warm climates selecting for a low pathogen growth rate. We also found a mirror effect of phenotypic plasticity and genetic adaptation on growth rate. At below the optimum, phenotypic plasticity enhances pathogen's growth rate but nature selects for slower growing genotypes when temperature increases. At above the optimum, phenotypic plasticity reduces pathogen's growth rate but natural selection favours for faster growing genotypes when temperature increases further. We conclude from these findings that the growth rate of P. infestans will only be marginally affected by global warming.

Diverse mechanisms shape the evolution of virulence factors in the potato late blight pathogen Phytophthora infestans sampled from China.

Evolution of virulence in plant pathogens is still poorly understood but the knowledge is important for the effective use of plant resistance and sustainable disease management. Spatial population dynamics of virulence, race and SSR markers in 140 genotypes sampled from seven geographic locations in China were compared to infer the mechanisms driving the evolution of virulence in Phytophthora infestans (P. infestans). All virulence types and a full spectrum of race complexity, ranging from the race able to infect the universally susceptible cultivar only to all differentials, were detected. Eight and two virulence factors were under diversifying and constraining selection respectively while no natural selection was detected in one of the virulence types. Further analyses revealed excesses in simple and complex races but deficiency in intermediate race and negative associations of annual mean temperature at the site from which pathogen isolates were collected with frequency of virulence to differentials and race complexity in the pathogen populations. These results suggest that host selection may interact with other factors such as climatic conditions in determining the evolutionary trajectory of virulence and race structure in P. infestans and global warming may slow down the emergence of new virulence in the pathogen.

Potato NPH3/RPT2-Like Protein StNRL1, Targeted by a Phytophthora infestans RXLR Effector, Is a Susceptibility Factor.

Plant pathogens deliver effectors to manipulate host processes. We know little about how fungal and oomycete effectors target host proteins to promote susceptibility, yet such knowledge is vital to understand crop disease. We show that either transient expression in Nicotiana benthamiana, or stable transgenic expression in potato (Solanum tuberosum), of the Phytophthora infestans RXLR effector Pi02860 enhances leaf colonization by the pathogen. Expression of Pi02860 also attenuates cell death triggered by the P. infestans microbe-associated molecular pattern INF1, indicating that the effector suppresses pattern-triggered immunity. However, the effector does not attenuate cell death triggered by Cf4/Avr4 coexpression, showing that it does not suppress all cell death activated by cell surface receptors. Pi02860 interacts in yeast two-hybrid assays with potato NPH3/RPT2-LIKE1 (NRL1), a predicted CULLIN3-associated ubiquitin E3 ligase. Interaction of Pi02860 in planta was confirmed by coimmunoprecipitation and bimolecular fluorescence complementation assays. Virus-induced gene silencing of NRL1 in N. benthamiana resulted in reduced P. infestans colonization and accelerated INF1-mediated cell death, indicating that this host protein acts as a negative regulator of immunity. Moreover, whereas NRL1 virus-induced gene silencing had no effect on the ability of the P. infestans effector Avr3a to suppress INF1-mediated cell death, such suppression by Pi02860 was significantly attenuated, indicating that this activity of Pi02860 is mediated by NRL1. Transient overexpression of NRL1 resulted in the suppression of INF1-mediated cell death and enhanced P. infestans leaf colonization, demonstrating that NRL1 acts as a susceptibility factor to promote late blight disease.

Comparative analyses of fungicide sensitivity and SSR marker variations indicate a low risk of developing azoxystrobin resistance in Phytophthora infestans.

Knowledge of the evolution of fungicide resistance is important in securing sustainable disease management in agricultural systems. In this study, we analyzed and compared the spatial distribution of genetic variation in azoxystrobin sensitivity and SSR markers in 140 Phytophthora infestans isolates sampled from seven geographic locations in China. Sensitivity to azoxystrobin and its genetic variation in the pathogen populations was measured by the relative growth rate (RGR) at four fungicide concentrations and determination of the effective concentration for 50% inhibition (EC50). We found that all isolates in the current study were sensitive to azoxystrobin and their EC50 was similar to that detected from a European population about 20 years ago, suggesting the risk of developing azoxystrobin resistance in P. infestans populations is low. Further analyses indicate that reduced genetic variation and high fitness cost in resistant mutations are the likely causes for the low evolutionary likelihood of developing azoxystrobin resistance in the pathogen. We also found a negative correlation between azoxystrobin tolerance in P. infestans populations and the mean annual temperature of collection sites, suggesting that global warming may increase the efficiency of using the fungicide to control the late blight.

[Comparative analysis of population genetic structure of Potato virus Y from different hosts].

Nucleotide sequences of P3 and pipo genes of Potato virus Y (PVY) from potato and tobacco were compared to investigate the effect of hosts on the population genetic structure. Meanwhile, mutation, natural selection and gene flow were evaluated to determine evolutionary forces responsible for the population genetic dynamics. The fixation indices of population differentiation (FST) of PVY from tobacco and potato were 0.116 and 0.120, respectively with significant difference, suggesting a moderate genetic differentiation between the two populations. Genetic variation analysis showed that nucleotide identities in P3 and pipo genes among the viral isolates from tobacco were respectively in the range of 85.2%-100% and 76.5%-100% while that from potato were respectively in the range of 95.7%-100% and 93.0%-100%, indicating higher genetic variation in PVY from tobacco than that from potato. Moreover, purifying selection was detected on the majority of polymorphic sites within P3 gene, suggesting that most of mutations in the gene were harmful and consequently being eliminated by natural selection. Conversely, positive selection was detected on two polymorphic sites, suggesting that these two mutations were beneficial to PVY. Neither purifying nor positive selection was detected in pipo gene, indicating neutral evolution of the gene. The values of gene flow (Nm) between PVY populations from tobacco and potato in P3 and pipo genes were 1.91 and 1.83, respectively, suggesting strong gene flow also contributes significantly to the population genetic dynamics of PVY population. In summary, this study indicates there was a significant genetic variation in PVY hosted by tobacco and potato, and mutation, natural selection and gene flow all contribute to the genetic diversity and population dynamic of the virus.

Sequence variation of P1 gene in Potato virus Y isolated from Fujian Province.

To understand the sequence variation and the putative protein structure of P1 gene in Potato virus Y (PVY) and to identify the sources of the variation, P1 gene in PVY isolated from Fujian Province was amplified by reverse-transcription polymerase chain reaction (RT-PCR) using a pair of degenerate primers designed from the conserved regions of published sequences. Sequence variation and putative protein structure were analyzed, and phylogenetic tree was reconstructed using Bayesian inference method. Expected fragments of 915 bp in size were amplified from 12 samples collected from Fujian Province by RT-PCR. The 12 sequences shared 73%-99% nucleotide identity with the reference sequences from GenBank. A strong recombination signal was identified at position 309 in sequences of isolates QK44, XT02, XT08 and LH05. Among the 12 sequences, 85 amino acid variants were detected, indicating high sequence variation in the P1 protein. However, positions 41-275 in the protein were highly conserved, especially in three active sites (H192, D201 and V235). Phylogenetic analysis grouped the sequences into three clades, each with different Coiled-coil domains and 3D-structures, suggesting divergent phylogenetic relationship among the groups. The above results show P1 gene in PVY is highly variable but contains 3 conserved active sites (H192, D201, V235) and the high genetic variation in the gene is primarily due to mutation and recombination.

[Sequence variation and protein structure of pipo gene in Potato virus Y].

The objectives of this study were to understand the sequence variation and the putative protein structure of pipo gene in the Potato virus Y (PVY) collected from Solanum tuberosum. The pipo gene in PVY was cloned using a pair of degenerate primers designed from its conserved region and its sequences were used to re-construct phylogenetic tree in Potyvirus genera by a Bayesian inference method. An expected fragment of 235 bp was amplified in all 20 samples by RT-PCR and the pipo genes in the 20 samples assayed shared more than 92% nucleotide sequence similarity with the published sequences of PVY strains. Among the 20 pipo gene sequences, 13 polymorphic sites were detected, including 4 parsimony informative sites and 9 singleton variable sites. These results indicate that PVY pipo gene is highly conserved but some sequence variations exist. Further analyses suggest that the pipo gene encodes a hydrophilic protein without signal peptide and transmembrane region. The protein has theoretical isoelectric points (pI) ranging from 11.26 to 11.62 and contains three highly conserved regions, especially between aa 10 and 59. The protein is likely located in the mitochondria and has a-helix secondary structure. Bayesian inference of phylogenetic trees reveals that PVY isolates are clustered in the same branch with high posterior probability, while Sunflower chlorotic mottle virus (SoCMoV) and Pepper severe mosaic virus (PepSMV) are closely related, consisting with the classification of Potyvirus genera using other approaches. Our analyses suggest that the pipo gene can be a new marker for phylogenetic analysis of the genera. The results reported in this paper provide useful insights in the genetic variation and the evolution of PVY and can stimulate further research on structure and function of the PIPO protein.