Duplication and neofunctionalization of a horizontally transferred xyloglucanase as a facet of the Red Queen coevolutionary dynamic.

Oomycete protists share phenotypic similarities with fungi, including the ability to cause plant diseases, but branch in a distant region of the tree of life. It has been suggested that multiple horizontal gene transfers (HGTs) from fungi-to-oomycetes contributed to the evolution of plant-pathogenic traits. These HGTs are predicted to include secreted proteins that degrade plant cell walls, a barrier to pathogen invasion and a rich source of carbohydrates. Using a combination of phylogenomics and functional assays, we investigate the diversification of a horizontally transferred xyloglucanase gene family in the model oomycete species Phytophthora sojae. Our analyses detect 11 xyloglucanase paralogs retained in P. sojae. Using heterologous expression in yeast, we show consistent evidence that eight of these paralogs have xyloglucanase function, including variants with distinct protein characteristics, such as a long-disordered C-terminal extension that can increase xyloglucanase activity. The functional variants analyzed subtend a phylogenetic node close to the fungi-to-oomycete transfer, suggesting the horizontally transferred gene was a bona fide xyloglucanase. Expression of three xyloglucanase paralogs in Nicotiana benthamiana triggers high-reactive oxygen species (ROS) generation, while others inhibit ROS responses to bacterial immunogens, demonstrating that the paralogs differentially stimulate pattern-triggered immunity. Mass spectrometry of detectable enzymatic products demonstrates that some paralogs catalyze the production of variant breakdown profiles, suggesting that secretion of variant xyloglucanases increases efficiency of xyloglucan breakdown as well as diversifying the damage-associated molecular patterns released. We suggest that this pattern of neofunctionalization and the variant host responses represent an aspect of the Red Queen host-pathogen coevolutionary dynamic.

Identification of Phytophthora cinnamomi CRN effectors and their roles in manipulating cell death during Persea americana infection.

The oomycete Phytophthora cinnamomi is a devastating plant pathogen with a notably broad host range. It is the causal agent of Phytophthora root rot (PRR), arguably the most economically important yield-limiting disease in Persea americana (avocado). Despite this, our understanding of the mechanisms P. cinnamomi employs to infect and successfully colonize avocado remains limited, particularly regarding the pathogen's ability to maintain its biotrophic and necrotrophic lifestyles during infection. The pathogen utilises a large repertoire of effector proteins which function in facilitating and establishing disease in susceptible host plants. Crinkling and necrosis effectors (CRN/Crinklers) are suspected to manipulate cell death to aid in maintenance of the pathogens biotrophic and necrotrophic lifestyles during different stages of infection. The current study identified 25 P. cinnamomi CRN effectors from the GKB4 genome using an HMM profile and assigned putative function to them as either cell death inducers or suppressors. Function was assigned to 10 PcinCRNs by analysing their RNA-seq expression profiles, relatedness to other functionally characterised Phytophthora CRNs and tertiary protein predictions. The full-length coding sequences for these PcinCRNs were confirmed by Sanger sequencing, six of which were found to have two divergent alleles. The presence of alleles indicates that the proteins encoded may perform contradicting functions in cell death manipulation, or function in different host plant species. Overall, this study provides a foundation for future research on P. cinnamomi infection and cell death manipulation mechanisms.

Insights into the genetic architecture of Phytophthora capsici root rot resistance in chile pepper (Capsicum spp.) from multi-locus genome-wide association study.

BACKGROUND: Phytophthora root rot, a major constraint in chile pepper production worldwide, is caused by the soil-borne oomycete, Phytophthora capsici. This study aimed to detect significant regions in the Capsicum genome linked to Phytophthora root rot resistance using a panel consisting of 157 Capsicum spp. genotypes. Multi-locus genome wide association study (GWAS) was conducted using single nucleotide polymorphism (SNP) markers derived from genotyping-by-sequencing (GBS). Individual plants were separately inoculated with P. capsici isolates, 'PWB-185', 'PWB-186', and '6347', at the 4-8 leaf stage and were scored for disease symptoms up to 14-days post-inoculation. Disease scores were used to calculate disease parameters including disease severity index percentage, percent of resistant plants, area under disease progress curve, and estimated marginal means for each genotype. RESULTS: Most of the genotypes displayed root rot symptoms, whereas five accessions were completely resistant to all the isolates and displayed no symptoms of infection. A total of 55,117 SNP markers derived from GBS were used to perform multi-locus GWAS which identified 330 significant SNP markers associated with disease resistance. Of these, 56 SNP markers distributed across all the 12 chromosomes were common across the isolates, indicating association with more durable resistance. Candidate genes including nucleotide-binding site leucine-rich repeat (NBS-LRR), systemic acquired resistance (SAR8.2), and receptor-like kinase (RLKs), were identified within 0.5 Mb of the associated markers. CONCLUSIONS: Results will be used to improve resistance to Phytophthora root rot in chile pepper by the development of Kompetitive allele-specific markers (KASP®) for marker validation, genomewide selection, and marker-assisted breeding.

CDC48 regulates immunity pathway in tobacco plants.

The CDC48 protein, highly conserved in the living kingdom, is a player of the ubiquitin proteasome system and contributes to various cellular processes. In plants, CDC48 is involved in cell division, plant growth and, as recently highlighted in several reports, in plant immunity. In the present study, to further extend our knowledge about CDC48 functions in plants, we analysed the incidence of its overexpression on tobacco development and immune responses. CDC48 overexpression disrupted plant development and morphology, induced changes in plastoglobule appearance and exacerbated ROS production. In addition, levels of salicylic acid (SA) and glycosylated SA were higher in transgenic plants, both in the basal state and in response to cryptogein, a protein produced by the oomycete Phytophthora cryptogea triggering defence responses. The expression of defence genes, notably those coding for some pathogenesis-related (PR) proteins, was also exacerbated in the basal state in transgenic plant lines. Finally, tobacco plants overexpressing CDC48 did not develop necrosis in response to tobacco mosaic virus (TMV) infection, suggesting a role for CDC48 in virus resistance.

A MYB-related transcription factor regulates effector gene expression in an oomycete pathogen.

Phytophthora pathogens possess hundreds of effector genes that exhibit diverse expression patterns during infection, yet how the expression of effector genes is precisely regulated remains largely elusive. Previous studies have identified a few potential conserved transcription factor binding sites (TFBSs) in the promoters of Phytophthora effector genes. Here, we report a MYB-related protein, PsMyb37, in Phytophthora sojae, the major causal agent of root and stem rot in soybean. Yeast one-hybrid and electrophoretic mobility shift assays showed that PsMyb37 binds to the TACATGTA motif, the most prevalent TFBS in effector gene promoters. The knockout mutant of PsMyb37 exhibited significantly reduced virulence on soybean and was more sensitive to oxidative stress. Consistently, transcriptome analysis showed that numerous effector genes associated with suppressing plant immunity or scavenging reactive oxygen species were down-regulated in the PsMyb37 knockout mutant during infection compared to the wild-type P. sojae. Several promoters of effector genes were confirmed to drive the expression of luciferase in a reporter assay. These results demonstrate that a MYB-related transcription factor contributes to the expression of effector genes in P. sojae.

Receptor-like cytoplasmic kinases of different subfamilies differentially regulate SOBIR1/BAK1-mediated immune responses in Nicotiana benthamiana.

Cell-surface receptors form the front line of plant immunity. The leucine-rich repeat (LRR)-receptor-like kinases SOBIR1 and BAK1 are required for the functionality of the tomato LRR-receptor-like protein Cf-4, which detects the secreted effector Avr4 of the pathogenic fungus Fulvia fulva. Here, we show that the kinase domains of SOBIR1 and BAK1 directly phosphorylate each other and that residues Thr522 and Tyr469 of the kinase domain of Nicotiana benthamiana SOBIR1 are required for its kinase activity and for interacting with signalling partners, respectively. By knocking out multiple genes belonging to different receptor-like cytoplasmic kinase (RLCK)-VII subfamilies in N. benthamiana:Cf-4, we show that members of RLCK-VII-6, -7, and -8 differentially regulate the Avr4/Cf-4-triggered biphasic burst of reactive oxygen species. In addition, members of RLCK-VII-7 play an essential role in resistance against the oomycete pathogen Phytophthora palmivora. Our study provides molecular evidence for the specific roles of RLCKs downstream of SOBIR1/BAK1-containing immune complexes.

Complete telomere-to-telomere genomes uncover virulence evolution conferred by chromosome fusion in oomycete plant pathogens.

Variations in chromosome number are occasionally observed among oomycetes, a group that includes many plant pathogens, but the emergence of such variations and their effects on genome and virulence evolution remain ambiguous. We generated complete telomere-to-telomere genome assemblies for Phytophthora sojae, Globisporangium ultimum, Pythium oligandrum, and G. spinosum. Reconstructing the karyotype of the most recent common ancestor in Peronosporales revealed that frequent chromosome fusion and fission drove changes in chromosome number. Centromeres enriched with Copia-like transposons may contribute to chromosome fusion and fission events. Chromosome fusion facilitated the emergence of pathogenicity genes and their adaptive evolution. Effectors tended to duplicate in the sub-telomere regions of fused chromosomes, which exhibited evolutionary features distinct to the non-fused chromosomes. By integrating ancestral genomic dynamics and structural predictions, we have identified secreted Ankyrin repeat-containing proteins (ANKs) as a novel class of effectors in P. sojae. Phylogenetic analysis and experiments further revealed that ANK is a specifically expanded effector family in oomycetes. These results revealed chromosome dynamics in oomycete plant pathogens, and provided novel insights into karyotype and effector evolution.

Lactic acid induced defense responses in tobacco against Phytophthora nicotianae.

Induced resistance is considered an eco-friendly disease control strategy, which can enhance plant disease resistance by inducing the plant's immune system to activate the defense response. In recent years, studies have shown that lactic acid can play a role in plant defense against biological stress; however, whether lactic acid can improve tobacco resistance to Phytophthora nicotianae, and its molecular mechanism remains unclear. In our study, the mycelial growth and sporangium production of P. nicotianae were inhibited by lactic acid in vitro in a dose-dependent manner. Application of lactic acid could reduce the disease index, and the contents of total phenol, salicylic acid (SA), jasmonic acid (JA), lignin and H2O2, catalase (CAT) and phenylalanine ammonia-lyase (PAL) activities were significantly increased. To explore this lactic acid-induced protective mechanism for tobacco disease resistance, RNA-Seq analysis was used. Lactic acid enhances tobacco disease resistance by activating Ca2+, reactive oxygen species (ROS) signal transduction, regulating antioxidant enzymes, SA, JA, abscisic acid (ABA) and indole-3-acetic acid (IAA) signaling pathways, and up-regulating flavonoid biosynthesis-related genes. This study demonstrated that lactic acid might play a role in inducing resistance to tobacco black shank disease; the mechanism by which lactic acid induces disease resistance includes direct antifungal activity and inducing the host to produce direct and primed defenses. In conclusion, this study provided a theoretical basis for lactic acid-induced resistance and a new perspective for preventing and treating tobacco black shank disease.

A novel and ubiquitous miRNA-involved regulatory module ensures precise phosphorylation of RNA polymerase II and proper transcription.

Proper transcription orchestrated by RNA polymerase II (RNPII) is crucial for cellular development, which is rely on the phosphorylation state of RNPII's carboxyl-terminal domain (CTD). Sporangia, developed from mycelia, are essential for the destructive oomycetes Phytophthora, remarkable transcriptional changes are observed during the morphological transition. However, how these changes are rapidly triggered and their relationship with the versatile RNPII-CTD phosphorylation remain enigmatic. Herein, we found that Phytophthora capsici undergone an elevation of Ser5-phosphorylation in its uncanonical heptapeptide repeats of RNPII-CTD during sporangia development, which subsequently changed the chromosomal occupation of RNPII and primarily activated transcription of certain genes. A cyclin-dependent kinase, PcCDK7, was highly induced and phosphorylated RNPII-CTD during this morphological transition. Mechanistically, a novel DCL1-dependent microRNA, pcamiR1, was found to be a feedback modulator for the precise phosphorylation of RNPII-CTD by complexing with PcAGO1 and regulating the accumulation of PcCDK7. Moreover, this study revealed that the pcamiR1-CDK7-RNPII regulatory module is evolutionarily conserved and the impairment of the balance between pcamiR1 and PcCDK7 could efficiently reduce growth and virulence of P. capsici. Collectively, this study uncovers a novel and evolutionary conserved mechanism of transcription regulation which could facilitate correct development and identifies pcamiR1 as a promising target for disease control.

The MYB Transcription Factor GmMYB78 Negatively Regulates Phytophthora sojae Resistance in Soybean.

Phytophthora root rot is a devastating disease of soybean caused by Phytophthora sojae. However, the resistance mechanism is not yet clear. Our previous studies have shown that GmAP2 enhances sensitivity to P. sojae in soybean, and GmMYB78 is downregulated in the transcriptome analysis of GmAP2-overexpressing transgenic hairy roots. Here, GmMYB78 was significantly induced by P. sojae in susceptible soybean, and the overexpressing of GmMYB78 enhanced sensitivity to the pathogen, while silencing GmMYB78 enhances resistance to P. sojae, indicating that GmMYB78 is a negative regulator of P. sojae. Moreover, the jasmonic acid (JA) content and JA synthesis gene GmAOS1 was highly upregulated in GmMYB78-silencing roots and highly downregulated in overexpressing ones, suggesting that GmMYB78 could respond to P. sojae through the JA signaling pathway. Furthermore, the expression of several pathogenesis-related genes was significantly lower in GmMYB78-overexpressing roots and higher in GmMYB78-silencing ones. Additionally, we screened and identified the upstream regulator GmbHLH122 and downstream target gene GmbZIP25 of GmMYB78. GmbHLH122 was highly induced by P. sojae and could inhibit GmMYB78 expression in resistant soybean, and GmMYB78 was highly expressed to activate downstream target gene GmbZIP25 transcription in susceptible soybean. In conclusion, our data reveal that GmMYB78 triggers soybean sensitivity to P. sojae by inhibiting the JA signaling pathway and the expression of pathogenesis-related genes or through the effects of the GmbHLH122-GmMYB78-GmbZIP25 cascade pathway.

Phase-specific transcriptional patterns of the oomycete pathogen Phytophthora sojae unravel genes essential for asexual development and pathogenic processes.

Oomycetes are filamentous microorganisms easily mistaken as fungi but vastly differ in physiology, biochemistry, and genetics. This commonly-held misconception lead to a reduced effectiveness by using conventional fungicides to control oomycetes, thus it demands the identification of novel functional genes as target for precisely design oomycetes-specific microbicide. The present study initially analyzed the available transcriptome data of the model oomycete pathogen, Phytophthora sojae, and constructed an expression matrix of 10,953 genes across the stages of asexual development and host infection. Hierarchical clustering, specificity, and diversity analyses revealed a more pronounced transcriptional plasticity during the stages of asexual development than that in host infection, which drew our attention by particularly focusing on transcripts in asexual development stage to eventually clustered them into 6 phase-specific expression modules. Three of which respectively possessing a serine/threonine phosphatase (PP2C) expressed during the mycelial and sporangium stages, a histidine kinase (HK) expressed during the zoospore and cyst stages, and a bZIP transcription factor (bZIP32) exclusive to the cyst germination stage were selected for down-stream functional validation. In this way, we demonstrated that PP2C, HK, and bZIP32 play significant roles in P. sojae asexual development and virulence. Thus, these findings provide a foundation for further gene functional annotation in oomycetes and crop disease management.

Genome-wide comprehensive analysis of miRNAs and their target genes expressed in resistant and susceptible Capsicum annuum genotypes during Phytophthora capsici infection.

Despite extensive works on miRNA's role during plant-oomycete interaction, its role in Capsicum annuum-Phytophthora capsici pathosystem is not fully explored. Therefore, the present study was designed to identify known and novel miRNAs along with their target genes in two contrasting chili peppers genotypes, i.e., GojamMecha_9086 (resistant) and Dabat_80045 (susceptible) under P. capsici infection associated with modulating the defense response during disease pathogenesis. The result demonstrated 79 known miRNAs corresponding to 24 miRNAs families and 477 novel miRNAs along with 22,895 potential targets, including 30 defense-related target genes against P. capsici infection. The expression analysis of 29 known and 157 novel miRNAs in resistant and 30 known and 177 novel miRNAs in susceptible genotypes revealed differential accumulation patterns. qRT-PCR analysis of 8 defense-related miRNAs representing 4 novels (Pz-novel-miR428-1, Pz-novel-miR160-1, Pz-novel-miR1028-1, Pz-novel-miR204-1) and 4 known miRNAs (Pz-known-miR803-1, Pz-known-miR2059-1, Pz-known-miR2560-1, Pz-known-miR1872-1) revealed differential accumulation pattern in both resistant and susceptible genotypes. Additionally, validation of eight target genes of miRNAs using regional amplification quantitative RT-PCR (RA-PCR), a superior technique to 5'-RNA Ligase-Mediated-rapid amplification of cDNA ends (5' RLM-RACE), revealed expression of six target genes positively correlated with their corresponding miRNAs in RC versus RI leaf, while five target genes observed an inverse correlation with their corresponding miRNAs in SC versus SI leaf, suggesting their key role during disease response. The Pz-known-miR1872-PODs pair showed perfect inverse relation in all four samples. The significant findings of the current study provide comprehensive genome-wide information about the repertoire of miRNAs and their target genes expressed in resistant and susceptible chili pepper genotypes, which can serve as a valuable resource for better understanding the post-transcriptional regulatory mechanism during C. annuum-P. capsici pathosystem.

Development of Screening Assays for Pathogen Virulence and Resistance to Phytophthora Root Rot and Wilting Complex in Raspberry.

Phytophthora root rot and wilting complex (PRRW) of red raspberry, caused primarily by Phytophthora rubi, is an economically important disease in British Columbia (BC) and in raspberry producing regions globally. Reliable, rapid, and efficient screening methods are lacking for evaluating germplasm for potential disease resistance in raspberry breeding programs as well as for screening pathogen isolates for virulence. The objective of this study was to compare various screening methods for efficiency and rapidity in inducing symptoms of disease to identify the most suitable approach. We compared several intact plant root inoculation (IPRI) assays, detached stem assays, and an intact plant stem inoculation (IPSI) assay. A virulent isolate of P. rubi was inoculated in two commercial cultivars: 'Chemainus' (susceptible to PRRW) and 'Cascade Bounty' (moderately resistant to PRRW). For IPRI assays, days to first symptom development, plant wilt progression, and root assessment were recorded. For detached stem tissue and IPSI assays, days to first visible lesions and lesion size were assessed. Experiments were arranged in a completely randomized design with three replications in each experiment. Three IPRI assays produced reliable symptoms in both cultivars. Among the detached stem assays, a node inoculation method performed better than other methods. Detached stem assays are useful for rapid pathogenicity testing of P. rubi, whereas IPRI assays are better for screening germplasm for disease resistance. Overall, this study identified several assays that can be used for conducting studies on pathogen phenotypic diversity (pathogenicity and virulence tests) and screening raspberry cultivars, germplasm, and breeding materials for response to PRRW.

Autophagy-Related Gene PlATG6a Is Involved in Mycelial Growth, Asexual Reproduction and Tolerance to Salt and Oxidative Stresses in Peronophythora litchii.

Autophagy is ubiquitously present in eukaryotes. During this process, intracellular proteins and some waste organelles are transported into lysosomes or vacuoles for degradation, which can be reused by the cell to guarantee normal cellular metabolism. However, the function of autophagy-related (ATG) proteins in oomycetes is rarely known. In this study, we identified an autophagy-related gene, PlATG6a, encoding a 514-amino-acid protein in Peronophythora litchii, which is the most destructive pathogen of litchi. The transcriptional level of PlATG6a was relatively higher in mycelium, sporangia, zoospores and cysts. We generated PlATG6a knockout mutants using CRISPR/Cas9 technology. The P. litchii Δplatg6a mutants were significantly impaired in autophagy and vegetative growth. We further found that the Δplatg6a mutants displayed decreased branches of sporangiophore, leading to impaired sporangium production. PlATG6a is also involved in resistance to oxidative and salt stresses, but not in sexual reproduction. The transcription of peroxidase-encoding genes was down-regulated in Δplatg6a mutants, which is likely responsible for hypersensitivity to oxidative stress. Compared with the wild-type strain, the Δplatg6a mutants showed reduced virulence when inoculated on the litchi leaves using mycelia plugs. Overall, these results suggest a critical role for PlATG6a in autophagy, vegetative growth, sporangium production, sporangiophore development, zoospore release, pathogenesis and tolerance to salt and oxidative stresses in P. litchii.

Local Eradication of Phytophthora ramorum Is Effective on Both NA1 and EU1 Lineages in Oregon Tanoak Forests.

Sudden oak death (SOD), caused by the oomycete Phytophthora ramorum, has been actively managed in Oregon since its discovery there in 2001. SOD is a devastating disease affecting an ecologically and culturally important tree species in southwestern Oregon. Initially infested with the NA1 lineage, the more virulent EU1 lineage was discovered in 2015. Management has adapted over time in response to experimental findings and administrative limitations. Current management practices present an opportunity to compare the efficacy of treatment on these lineages by analyzing P. ramorum inoculum at untreated and treated sites. Current treatment includes herbicide treatment on host stems followed by felling, piling, and burning on site. Infested sites were visited between 2018 and 2020 (n = 88), where understory vegetation and soil was collected. Generalized linear modeling demonstrated that treatment had a significant impact on P. ramorum prevalence from vegetation samples, with an average of 33% (± 10%) fewer positive samples at treated sites. Linear mixed-effects modeling of a subpopulation of EU1 sites visited before and after treatment showed a similar effect of treatment, with a 43% (± 15%) reduction in P. ramorum prevalence. Prevalence of P. ramorum in soil was not affected by treatment in either analysis. A third analysis taking into consideration recent wildfire incursion into infested areas revealed that wildfire alone is insufficient to reduce prevalence of P. ramorum. These results strongly suggest that management is successfully reducing P. ramorum inoculum found on understory vegetation, and that treatment remains necessary to reduce the spread of this major forest pathogen.

In silico characterization of molecular factors involved in metabolism and pathogenicity of Phytophthora cinnamomi.

Phytophthora cinnamomi is classified as one of the most devastating plant pathogens in the world. It has a destructive effect on more than 5000 horticultural and forestry species in the world, and especially on Castanea sativa. The genus Phytophthora belongs to the Class Oomycetes, a group of fungus like organisms which provoke plant diseases via motile zoospores. Control of this organism is considered very challenging because of the limited range of effective chemical inhibitors. The development of sustainable control measures for the future management of P. cinnamomi requires in-depth knowledge of the cellular and molecular bases of development and metabolism. The aim of this review was to identify molecular factors associated with the metabolism of P. cinnamomi by studying the genes implicated in fundamental metabolism using tools of bioinformatics. Also, some genes involved in pathogenicity will be cited and characterized, such as genes coding for transglycosylases. Genomic sequences of P. cinnamomi were analyzed using an open reading frame (ORF) finder. The identified ORFs products (proteins) were compared to sequences already described and with known functions present in databases such as NCBI and fungi database. In this way, homologous proteins were found, with the respective specific domains, to proteins involved in the metabolism and pathogenicity of Phytophthora ssp.

A giant NLR gene confers broad-spectrum resistance to Phytophthora sojae in soybean.

Phytophthora root and stem rot caused by P. sojae is a destructive soybean soil-borne disease found worldwide. Discovery of genes conferring broad-spectrum resistance to the pathogen is a need to prevent the outbreak of the disease. Here, we show that soybean Rps11 is a 27.7-kb nucleotide-binding site-leucine-rich repeat (NBS-LRR or NLR) gene conferring broad-spectrum resistance to the pathogen. Rps11 is located in a genomic region harboring a cluster of large NLR genes of a single origin in soybean, and is derived from rounds of unequal recombination. Such events result in promoter fusion and LRR expansion that may contribute to the broad resistance spectrum. The NLR gene cluster exhibits drastic structural diversification among phylogenetically representative varieties, including gene copy number variation ranging from five to 23 copies, and absence of allelic copies of Rps11 in any of the non-Rps11-donor varieties examined, exemplifying innovative evolution of NLR genes and NLR gene clusters.

Epigenetic responses to Phytophthora citrophthora gummosis in citrus.

Studies show that DNA methylation is associated with plant immunity but little is known as to how this epigenetic mechanism assists plants in adjusting their responses to biotic stress, especially when interacting with an hemibiotrophic pathogen such as citrus Phytophthora. The aim of the present study was to assess the effects of scion-rootstock interaction on plant resistance to P. citrophthora infection and DNA methylation patterns in 'Pera' sweet orange and 'Tahiti' acid lime grafted onto 'Rangpur' lime and 'Tropical' sunki rootstocks reinoculated with P. citrophthora. Results showed that reinoculated plants of the 'Pera' sweet orange/'Rangpur' lime and 'Tahiti' acid lime/'Tropical' sunki combinations with more and less sensitive varieties to Phytophthora, presented smaller stem lesions and increased frequency of full methylation and hemimethylation rates, compared to inoculated plants. In contrast, 'Tahiti' acid lime/'Rangpur' lime, two highly sensitive varieties, and 'Pera'/'Tropical' sunki, two much less sensitive varieties, showed high increases in the frequency of hemimethylation and non-methylation levels. Results suggest that in citrus, both the scion-rootstock interaction and DNA methylation affect the response to P. citrophthora infection. Reinoculated plants, depending on the combination, showed changes in intracellular hyphae growth through the formation of sets of fibers and crystal accumulation in the periderm, cortex, and phloem. In addition, starch grain concentration was higher in reinoculated plants in comparison to inoculated plants. These findings support the assumption that DNA methylation is a plant defense mechanism and therefore may be exploited to improve the response of plants to the gummosis of P. citrophthora in citrus.

Detecting Phytophthora cinnamomi associated with dieback disease on Carya cathayensis using loop-mediated isothermal amplification.

Chinese hickory (Carya cathayensis Sarg.) is an economically and ecologically important nut plant in China. Dieback and basal stem necrosis have been observed in the plants since 2016, and its recent spread has significantly affected plant growth and nut production. Therefore, a survey was conducted to evaluate the disease incidence at five sites in Linan County, China. The highest incidence was recorded at the Tuankou site at up to 11.39% in 2019. The oomycete, Phytophthora cinnamomi, was isolated from symptomatic plant tissue and plantation soil using baiting and selective media-based detection methods and identified. Artificial infection with the representative P. cinnamomi ST402 isolate produced vertically elongated discolorations in the outer xylem and necrotic symptoms in C. cathayensis seedlings in a greenhouse trial. Molecular detections based on loop-mediated isothermal amplification (LAMP) specific to P. cinnamomi ST402 were conducted. Result indicated that LAMP detection showed a high coherence level with the baiting assays for P. cinnamomi detection in the field. This study provides the evidence of existence of high-pathogenic P. cinnamomi in the C. cathayensis plantation soil in China and the insights into a convenient tool developed for conducting field monitoring of this aggressive pathogen.

The Plant-Beneficial Rhizobacterium Bacillus velezensis FZB42 Controls the Soybean Pathogen Phytophthora sojae Due to Bacilysin Production.

Soybean root rot caused by the oomycete Phytophthora sojae is a serious soilborne disease threatening soybean production in China. Bacillus velezensis FZB42 is a model strain for Gram-positive plant growth-promoting rhizobacteria and is able to produce multiple antibiotics. In this study, we demonstrated that B. velezensis FZB42 can efficiently antagonize P. sojae. The underlying mechanism for the inhibition was then investigated. The FZB42 mutants deficient in the synthesis of lipopeptides (bacillomycin D and fengycin), known to have antifungal activities, and polyketides (bacillaene, difficidin, and macrolactin), known to have antibacterial activities, were not impaired in their antagonism toward P. sojae; in contrast, mutants deficient in bacilysin biosynthesis completely lost their antagonistic activities toward P. sojae, indicating that bacilysin was responsible for the activity. Isolated pure bacilysin confirmed this inference. Bacilysin was previously shown to be antagonistic mainly toward prokaryotic bacteria rather than eukaryotes. Here, we found that bacilysin could severely damage the hyphal structures of P. sojae and lead to the loss of its intracellular contents. A device was invented allowing interactions between P. sojae and B. velezensis FZB42 on nutrient agar. In this manner, the effect of FZB42 on P. sojae was studied by transcriptomics. FZB42 significantly inhibited the expression of P. sojae genes related to growth, macromolecule biosynthesis, pathogenicity, and ribosomes. Among them, the genes for pectate lyase were the most significantly downregulated. Additionally, we showed that bacilysin effectively prevents soybean sprouts from being infected by P. sojae and could antagonize diverse Phytophthora species, such as Phytophthora palmivora, P. melonis, P. capsici, P. litchi, and, most importantly, P. infestans. IMPORTANCEPhytophthora spp. are widespread eukaryotic phytopathogens and often extremely harmful. Phytophthora can infect many types of plants important to agriculture and forestry and thus cause large economic losses. Perhaps due to inappropriate recognition of Phytophthora as a common pathogen in history, research on the biological control of Phytophthora is limited. This study shows that B. velezensis FZB42 can antagonize various Phytophthora species and prevent the infection of soybean seedlings by P. sojae. The antibiotic produced by FZB42, bacilysin, which was already known to have antibacterial effectiveness, is responsible for the inhibitory action against Phytophthora. We further showed that some Phytophthora genes and pathways may be targeted in future biocontrol studies. Therefore, our data provide a basis for the development of new tools for the prevention and control of root and stem rot in soybean and other plant diseases caused by Phytophthora.