Gene Expression in Cucurbita spp. Root and Crown during Phytophthora capsici Infection

Phytophtora capsici causes major diseases in cucurbit crops worldwide. In this study, we inoculated this pathogen into Cucurbita pepo subsp. pepo susceptible MUCU-16 and C. moschata tolerant M63. The gene expression of plant pathogenesis-related proteins chitinase (CpChiIV), lignin-forming peroxidase (CpLPOX), and defensin (CpDEF) and hormone-related enzymes salicylic acid (CpPAL) and ethylene (CpACO) was analyzed for two weeks post-inoculation in root and crown tissues. Differentially expressed genes were found between genotypes, tissues, days post-inoculation, and inoculated/non-inoculated samples. After inoculation, CpPAL and CpChiIV (crown) were downregulated in MUCU-16, while CpLPOX and CpDEF were upregulated in M63. In inoculated samples, higher expression changes were presented on days 10–14 than on day 3 for CpACO, CpLPOX, and CpDEF genes. Overexpression was higher for CpDEF compared to the other tested genes, indicating good suitability as a marker of biotic stress. The overexpression of CpDEF was higher in crown than in roots for both inoculated genotypes. The basal expression of CpPAL and CpDEF was higher in MUCU-16, but after inoculation, CpPAL and CpDEF gene expression were higher in M63. These changes suggest an association between CpDEF upregulation and tolerance, and between CpPAL downregulation and susceptibility.

Extracellular Vesicles from Fusarium graminearum Contain Protein Effectors Expressed during Infection of Corn

Fusarium graminearum (Fgr) is a devastating filamentous fungal pathogen that causes diseases in cereals, while producing mycotoxins that are toxic for humans and animals, and render grains unusable. Low efficiency in managing Fgr poses a constant need for identifying novel control mechanisms. Evidence that fungal extracellular vesicles (EVs) from pathogenic yeast have a role in human disease led us to question whether this is also true for fungal plant pathogens. We separated EVs from Fgr and performed a proteomic analysis to determine if EVs carry proteins with potential roles in pathogenesis. We revealed that protein effectors, which are crucial for fungal virulence, were detected in EV preparations and some of them did not contain predicted secretion signals. Furthermore, a transcriptomic analysis of corn (Zea mays) plants infected by Fgr revealed that the genes of some of the effectors were highly expressed in vivo, suggesting that the Fgr EVs are a mechanism for the unconventional secretion of effectors and virulence factors. Our results expand the knowledge on fungal EVs in plant pathogenesis and cross-kingdom communication, and may contribute to the discovery of new antifungals.

NorA, HmpX, and NorB cooperate to reduce NO toxicity during denitrification and plant pathogenesis in Ralstonia solanacearum

Ralstonia solanacearum, which causes bacterial wilt disease of many crops, needs denitrifying respiration to succeed in hypoxic plant xylem vessels. Inside its host this pathogen confronts toxic oxidative radicals like nitric oxide (NO) generated by both bacterial denitrification and host defenses. R. solanacearum has multiple distinct mechanisms that could mitigate this stress, including Repair of Iron Cluster (RIC) homolog NorA, nitric oxide reductase NorB, and flavohaemoglobin HmpX. R. solanacearum upregulated norA, norB, and hmpX in response to exogenous NO, denitrification, and tomato pathogenesis. Single mutants lacking any of these genes accumulated NO during denitrification and were more susceptible to oxidative stress. Plant defense genes were upregulated in tomatoes infected with the NO-overproducing ΔnorB mutant, suggesting bacterial detoxification of NO reduces pathogen visibility. Expression of many iron and sulfur metabolism genes increased in the ΔnorB, ΔnorA, and ΔhmpX mutants, suggesting that losing even one NO detoxification system demands metabolic compensation. Single mutants suffered only moderate fitness reductions in host plants, possibly because they upregulated their remaining detoxification genes. However, ΔnorA/norB, ΔnorB/hmpX, and ΔnorA/hmpX double mutants grew poorly in denitrifying culture and in planta. Loss of norA, norB, and hmpX may be lethal as the methods used to construct the double mutants did not generate a triple mutant. Aconitase activity assays showed that NorA, HmpX and especially NorB are important for maintaining iron-sulfur cluster proteins. Thus, R. solanacearum's three NO detoxification systems each contribute to and are collectively essential for overcoming oxidative stress during denitrification and growth in a host plant.

Assessment of Inoculation Methods of Thielaviopsis paradoxa (De Seynes) Höhn into Oil Palm Seedlings under Greenhouse Conditions

Oil palm (Elaeis guineensis Jacq. and Elaeis Oleifera Cortes) is one of the most important oil crops in the world. Colombia is the fourth-largest oil palm producer worldwide. However, oil palm diseases are a significant factor affecting yield. Thielaviopsis paradoxa (De Seynes) Höhn is a pathogen that affects young palm trees, causing spear rot. Four disease establishment methods were studied to replicate, in a controlled environment, the symptoms of the disease found in the field. Young palm trees were inoculated with a suspension of endoconidia using either local infiltration, drip, scissor cut, or direct contact with agar blocks bearing mycelia and conidia. The effects of the inoculation methods were studied in dose-method-disease severity experiments conducted in a greenhouse under controlled conditions. All four methods resulted in T. paradoxa infections and the development of symptoms of the disease. The disease severity was correlated with the method and dose of inoculation. In trials to test Koch’s postulates, T. paradoxa was isolated from areas of disease progression in the inoculated trees, but the teleomorph Ceratocystis paradoxa (Dade) Moreau was not observed. A photographic record of the infection process at different times post-infection was compiled. Given that establishing the disease through artificial inoculation is essential for assessing plant pathogenesis, this study determined that the local infiltration method (1 × 106 endoconidia mL−1) and a 3–7 day incubation period were critical for the development of symptoms as severe as those observed in natural infections in the field.

Long-term maize-Desmodium intercropping shifts structure and composition of soil microbiome with stronger impact on fungal communities

Purpose Push–pull is an intercropping technology that is rapidly spreading among smallholder farmers in Sub-Saharan Africa. The technology intercrops cereals with Desmodium to fight off stem borers, eliminate parasitic weeds, and improve soil fertility and yields of cereals. The above-ground components of push–pull cropping have been well investigated. However, the impact of the technology on the soil microbiome and the subsequent role of the microbiome on diverse ecosystem benefits are unknown. Here we describe the soil microbiome associated with maize—Desmodium intercropping in push–pull farming in comparison to long-term maize monoculture. Methods Soil samples were collected from long-term maize—Desmodium intercropping and maize monoculture plots at the international centre for insect physiology and ecology (ICIPE), Mbita, Kenya. Total DNA was extracted before16S rDNA and ITS sequencing and subsequent analysis on QIIME2 and R. Results Maize—Desmodium intercropping caused a strong divergence in the fungal microbiome, which was more diverse and species rich than monoculture plots. Fungal groups enriched in intercropping plots are linked to important ecosystem services, belonging to functional groups such as mycorrhiza, endophytes, saprophytes, decomposers and bioprotective fungi. Fewer fungal genera were enriched in monoculture plots, some of which were associated with plant pathogenesis and opportunistic infection in humans. In contrast, the impact of intercropping on soil bacterial communities was weak with few differences between intercropping and monoculture. Conclusion Maize—Desmodium intercropping diversifies fungal microbiomes and favors taxa associated with important ecosystem services including plant health, productivity and food safety.

High-Throughput SSR Marker Development and the Analysis of Genetic Diversity in Capsicum frutescens

Capsicum frutescens, one of the domesticated species of pepper grown worldwide, is thought to be highly advantageous due to its strong resistance against plant pathogenesis, high productivity, and intense aroma. However, a shortage of molecular markers limits the efficiency and accuracy of genetic breeding for pepper. With the newly developed next-generation sequencing technology, genome sequences of C. frutescens can be generated, which are now available for identifying SSR markers via data mining. In this study, a total of 278,425 SSRs were detected from the pepper genome using MISA software. It was observed that trinucleotides were the dominant repeat motif. This was followed by dinucleotides, tetranucleotides, pentanucleotides, and the hexanucleotides repeat types. (AT)n (TTG)n (AAAT)n (AAATA)n (TATAGA)n is known to be the most common repeat motifs corresponding to dinucleotide to hexanucleotide repeats, respectively. In addition, a total of 240 SSR primers evenly distributed over all 12 chromosomes were designed and screened against 8 C. frutescens cultivars. Of these, 33 SSR markers that have high polymorphism, have been scrutinized for 147 accessions from 25 countries. The dendrogram constructed clustered these accessions into seven major groups. The groups were found to be consistent with their origins. The results obtained in this study provided resources of SSR molecular markers and insight into genetic diversity of the C. frutescens.

Pseudomonas syringae addresses distinct environmental challenges during plant infection through the coordinated deployment of polysaccharides

Prior to infection, phytopathogenic bacteria face a challenging environment on the plant surface, where they are exposed to nutrient starvation and abiotic stresses. Pathways enabling surface adhesion, stress tolerance and epiphytic survival are important for successful plant pathogenesis. Understanding the roles and regulation of these pathways is therefore crucial to fully understand bacterial plant infections. The phytopathogen Pseudomonas syringae pv. tomato (Pst) encodes multiple polysaccharides that are implicated in biofilm formation, stress survival and virulence in other microbes. To examine how these polysaccharides impact Pst epiphytic survival and pathogenesis, we analysed mutants in multiple polysaccharide loci to determine their intersecting contributions to epiphytic survival and infection. In parallel, we used qRT-PCR to analyse the regulation of each pathway. Pst polysaccharides are tightly coordinated by multiple environmental signals. Nutrient availability, temperature and surface association strongly affect the expression of different polysaccharides under the control of the signalling proteins ladS and cbrB and the second messenger cyclic-di-GMP. Furthermore, functionally redundant, combinatorial phenotypes were observed for several polysaccharides. Exopolysaccharides and WapQ-mediated lipopolysaccharide production are important for leaf adhesion, while α-glucan and alginate together confer desiccation tolerance. Our results suggest that polysaccharides play important roles in overcoming environmental challenges to Pst during plant infection.

Adaptive evolution of Moniliophthora PR-1 proteins towards its pathogenic lifestyle

Background Plant pathogenesis related-1 (PR-1) proteins belong to the CAP superfamily and have been characterized as markers of induced defense against pathogens. Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that respectively cause the witches’ broom disease and frosty pod rot in Theobroma cacao. Interestingly, a large number of plant PR-1-like genes are present in the genomes of both species and many are up-regulated during the biotrophic interaction. In this study, we investigated the evolution of PR-1 proteins from 22 genomes of Moniliophthora isolates and 16 other Agaricales species, performing genomic investigation, phylogenetic reconstruction, positive selection search and gene expression analysis. Results Phylogenetic analysis revealed conserved PR-1 genes (PR-1a, b, d, j), shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora (PR-1f, g, h, i), as well as in recent specialization cases within M. perniciosa biotypes (PR-1c, k, l) and M. roreri (PR-1n). PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host–pathogen arms race. Furthermore, although previous work showed that MpPR-1 can detoxify plant antifungal compounds in yeast, we found that in the presence of eugenol M. perniciosa differentially expresses only MpPR-1e, k, d, of which two are not linked to pathogenicity, suggesting that detoxification might not be the main function of most MpPR-1. Conclusions Based on analyses of genomic and expression data, we provided evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Additionally, we also discuss how fungal PR-1 proteins could have adapted from basal conserved functions to possible roles in fungal pathogenesis.

The Cutinase Bdo_10846 Play an Important Role in the Virulence of Botryosphaeria dothidea and in Inducing the Wart Symptom on Apple Plant

Botryosphaeria dothidea is a pathogen with worldwide distribution, infecting hundreds of species of economically important woody plants. It infects and causes various symptoms on apple plants, including wart and canker on branches, twigs, and stems. However, the mechanism of warts formation is unclear. In this study, we investigated the mechanism of wart formation by observing the transection ultrastructure of the inoculated cortical tissues at various time points of the infection process and detecting the expression of genes related to the pathogen pathogenicity and plant defense response. Results revealed that wart induced by B. dothidea consisted of proliferous of phelloderm cells, the newly formed secondary phellem, and the suberized phelloderm cells surrounding the invading mycelia. The qRT-PCR analysis revealed the significant upregulation of apple pathogenesis-related and suberification-related genes and a pathogen cutinase gene Bdo_10846. The Bdo_10846 knockout transformants showed reduced cutinase activity and decreased virulence. Transient expression of Bdo_10846 in Nicotiana benthamiana induced ROS burst, callose formation, the resistance of N. benthamiana to Botrytis cinerea, and significant upregulation of the plant pathogenesis-related and suberification-related genes. Additionally, the enzyme activity is essential for the induction. Virus-induced gene silencing demonstrated that the NbBAK1 and NbSOBIR1 expression were required for the Bdo_10846 induced defense response in N. benthamiana. These results revealed the mechanism of wart formation induced by B. dothidea invasion and the important roles of the cutinase Bdo_10846 in pathogen virulence and in inducing plant immunity.