Fusarium Root Rot Complex in Soybean: Molecular Characterization, Trichothecene Formation, and Cross-Pathogenicity.

Soybean is threatened by many pathogens that negatively affect this crop's yield and quality, such as various Fusarium species that cause wilting and root rot diseases. Fusarium root rot (FRR) in soybean can be caused by F. graminearum and other Fusarium spp. that are associated with Fusarium head blight (FHB) in cereals. Therefore, it was important to inquire whether Fusarium pathogens from soybean can cause disease in wheat and vice versa. Here, we investigated the FRR complex in Manitoba (Canada) from symptomatic plants, using both culture- and molecular-based methods. We developed a molecular diagnostic toolkit to detect and differentiate between several Fusarium spp. involved in FHB and FRR, then we evaluated cross-pathogenicity of selected Fusarium isolates collected from soybean and wheat, and the results indicate that isolates recovered from one host can infect the other host. Trichothecene production by selected Fusarium spp. was also analyzed chemically via liquid chromatography mass spectrometry in both soybean (root) and wheat (spike) tissues. Trichothecenes were also analyzed in soybean seeds from plants with FRR to check the potentiality of trichothecene translocation from infected roots to the seeds. All of the tested Fusarium isolates were capable of producing trichothecenes in wheat spikes and soybean roots, but no trichothecenes were detected in soybean seeds. This study provided evidence, for the first time, that trichothecenes were produced by several Fusarium spp. (F. cerealis, F. culmorum, and F. sporotrichioides) during FRR development in soybean.

Gene Expression of Putative Pathogenicity-Related Genes in Verticillium dahliae in Response to Elicitation with Potato Extracts and during Infection Using Quantitative Real-Time PCR.

Quantitative real-time PCR was used to monitor the expression of 15 Verticillium dahliae's genes, putatively involved in pathogenicity, highly (HAV) and weakly aggressive (WAV) V. dahliae isolates after either (i) elicitation with potato leaf, stem, or root extracts, or (ii) inoculation of potato detached petioles. These genes, i.e., coding for Ras-GAP-like protein, serine/threonine protein kinase, Ubiquitin-conjugating enzyme variant-MMS2, NADH-ubiquinone oxidoreductase, Thioredoxin, Pyruvate dehydrogenase E1 VdPDHB, myo-inositol 2-dehydrogenase, and HAD-superfamily hydrolase, showed differential upregulation in the HAV versus WAV isolate in response to plant extracts or after inoculation of potato leaf petioles. This suggests their potential involvement in the observed differential aggressiveness between isolates. However, other genes like glucan endo-1,3-alpha-glucosidase and nuc-1 negative regulatory protein VdPREG showed higher activity in the WAV than in the HAV in response to potato extracts and/or during infection. This, in contrast, may suggest a role in their lower aggressiveness. These findings, along with future functional analysis of selected genes, will contribute to improving our understanding of V. dahliae's pathogenesis. For example, expression of VdPREG negatively regulates phosphorus-acquisition enzymes, which may indicate a lower phosphorus acquisition activity in the WAV. Therefore, integrating the knowledge about the activity of both genes enhancing pathogenicity and those restraining it will provide a guild line for further functional characterization of the most critical genes, thus driving new ideas towards better Verticillium wilt management.

MinION Nanopore-based detection of Clavibacter nebraskensis, the corn Goss's wilt pathogen, and bacteriomic profiling of necrotic lesions of naturally-infected leaf samples.

The Goss's bacterial wilt pathogen, Clavibacter nebraskensis, of corn is a candidate A1 quarantine organism; and its recent re-emergence and spread in the USA and Canada is a potential biothreat to the crop. We developed and tested an amplicon-based Nanopore detection system for C. nebraskensis (Cn), targeting a purine permease gene. The sensitivity (1 pg) of this system in mock bacterial communities (MBCs) spiked with serially diluted DNA of C. nebraskensis NCPPB 2581T is comparable to that of real-time PCR. Average Nanopore reads increased exponentially from 125 (1pg) to about 6000 reads (1000 pg) after a 3-hr run-time, with 99.0% of the reads accurately assigned to C. nebraskensis. Three run-times were used to process control MBCs, Cn-spiked MBCs, diseased and healthy leaf samples. The mean Nanopore reads doubled as the run-time is increased from 3 to 6 hrs while from 6 to 12 hrs, a 20% increment was recorded in all treatments. Cn-spiked MBCs and diseased corn leaf samples averaged read counts of 5,100, 11,000 and 14,000 for the respective run-times, with 99.8% of the reads taxonomically identified as C. nebraskensis. The control MBCs and healthy leaf samples had 47 and 14 Nanopore reads, respectively. 16S rRNA bacteriomic profiles showed that Sphingomonas (22.7%) and Clavibacter (21.2%) were dominant in diseased samples while Pseudomonas had only 3.5% relative abundance. In non-symptomatic leaf samples, however, Pseudomonas (20.0%) was dominant with Clavibacter at 0.08% relative abundance. This discrepancy in Pseudomonas abundance in the samples was corroborated by qPCR using EvaGreen chemistry. Our work outlines a new useful tool for diagnosis of the Goss's bacterial wilt disease; and provides the first insight on Pseudomonas community dynamics in necrotic leaf lesions.

Overexpression of Solanum tuberosum Respiratory Burst Oxidase Homolog A (StRbohA) Promotes Potato Tolerance to Phytophthora infestans.

Reactive oxygen species (ROSs) represent one of the first lines of plants' biochemical defense against pathogens. Plants' respiratory burst oxidase homologs (RBOHs) produce ROSs as byproducts in several cellular compartments. In potato tubers, Solanum tuberosum respiratory burst oxidase homolog (StRBOHs) are involved in suberization and healing of wounded tissues. StRbohA has been tested in the model plant Arabidopsis thaliana, which led to enhanced plant defense against the soilborne pathogen Verticillium dahliae. Here, we showed that overexpressing StRbohA in potato plants increases plant tolerance to the oomycete Phytophthora infestans, the causal agent of late blight disease. Transgenic potato plants expressing StRbohA showed reduced disease symptoms (necrosis) compared with the wild type. In parallel, the expression of pathogenesis-related genes (PRs); RBOHs; antioxidation-related genes CPRX1, PRX2, APRX1, CAT1, and CAT2; and genes involved in the biosynthesis pathways of jasmonic and salicylic acids (ICS, PAL1, PAL2, LOX1, LOX2, and LOX3) exhibited significant increases in transgenic plants in response to infection. After higher expression of RBOHs, ROSs accumulated more in inoculation sites of the transgenic plants. ROSs act as signals that activate gene expression in the salicylic acid (SA) biosynthesis pathway, leading to the accumulation of SA and triggering SA-based defense mechanisms. SA-responsive PRs showed higher expression in the transgenic plants, which resulted in the restriction of pathogen growth in plant tissues. These results demonstrate the effective role of StRbohA in increasing potato defense against P. infestans.

Specific Detection and Identification of Fusarium graminearum Sensu Stricto Using a PCR-RFLP Tool and Specific Primers Targeting the Translational Elongation Factor 1α Gene.

Fusarium graminearum is a toxigenic plant pathogen that causes Fusarium head blight (FHB) disease on cereal crops. It has recently shown to have cross-pathogenicity on noncereals (i.e., Fusarium root rot [FRR] on soybean) in Canada and elsewhere. Specific detection and differentiation of this potent toxigenic, trichothecene-producing pathogen among other closely related species is extremely important for disease control and mycotoxin monitoring. Here, we designed a PCR restriction fragment length polymorphism protocol based on the DNA sequence of the translational elongation factor 1α (TEF1α) gene. A unique restriction site to the enzyme HpaII is only found in F. graminearum sensu stricto strains among different Fusarium strains in the F. graminearum species complex (FGSC) and other Fusarium spp. associated with FHB in cereals and FRR in soybean. Partial amplification of the TEF1α gene with newly designed primers mh1/mh2 generated a 459-bp PCR fragment. Restriction digestion of the generated fragments with the HpaII enzyme generated a unique restriction pattern that can rapidly and accurately differentiate F. graminearum sensu stricto among all other Fusarium spp. A primer pair (FgssF/FgssR) specific to F. graminearum sensu stricto also was designed and can distinguish F. graminearum sensu stricto from all other Fusarium spp. in the FGSC and other closely related Fusarium spp. involved in FHB and FRR. This finding will be very useful for the specific detection of F. graminearum sensu stricto for diagnostic purposes as well as for the accurate detection of this pathogen in breeding and other research purposes.

Transcriptome Analysis of Rlm2-Mediated Host Immunity in the Brassica napus-Leptosphaeria maculans Pathosystem.

Our study investigated disease resistance in the Brassica napus-Leptosphaeria maculans pathosystem using a combination of laser microdissection, dual RNA sequencing, and physiological validations of large-scale gene sets. The use of laser microdissection improved pathogen detection and identified putative L. maculans effectors and lytic enzymes operative during host colonization. Within 24 h of inoculation, we detected large shifts in gene activity in resistant cotyledons associated with jasmonic acid and calcium signaling pathways that accelerated the plant defense response. Sequencing data were validated through the direct quantification of endogenous jasmonic acid levels. Additionally, resistance against L. maculans was abolished when the calcium chelator EGTA was applied to the inoculation site, providing physiological evidence of the role of calcium in B. napus immunity against L. maculans. We integrated gene expression data with all available information on cis-regulatory elements and transcription factor binding affinities to better understand the gene regulatory networks underpinning plant resistance to hemibiotrophic pathogens. These in silico analyses point to early cellular reprogramming during host immunity that are coordinated by CAMTA, BZIP, and bHLH transcription factors. Together, we provide compelling genetic and physiological evidence into the programming of plant resistance against fungal pathogens.

Verticillium dahliae's Isochorismatase Hydrolase Is a Virulence Factor That Contributes to Interference With Potato's Salicylate and Jasmonate Defense Signaling.

This study aimed to dissect the function of the Isochorismatase Hydrolase (ICSH1) gene in Verticillium dahliae's pathogenesis on potato. VdICSH1 was up-regulated in V. dahliae after induction with extracts from potato tissues. Its expression increased more in response to root extracts than to leaf and stem extracts. However, such expression in response to root extracts was not significantly different in the highly and weakly aggressive isolates tested. During infection of detached potato leaves, VdICSH1 expression increased significantly in the highly aggressive isolate compared to the weakly aggressive one. We generated icsh1 mutants from a highly aggressive isolate of V. dahliae and compared their pathogenicity with that of the original wild type strain. The analysis showed that this gene is required for full virulence of V. dahliae on potatoes. When we previously found differential accumulation of ICSH1 protein in favor of the highly aggressive isolate, as opposed to the weakly aggressive one, we had hypothesized that ICSH would interfere with the host's defense SA-based signaling. Here, we measured the accumulation of both salicylic acid (SA) and jasmonic acid (JA) in potato plants inoculated with an icsh1 mutant in comparison with the wild type strain. The higher accumulation of bound SA in the leaves in response to the icsh1 mutant compared to the wild type confirms the hypothesis that ICSH1 interferes with SA. However, the different trends in SA and JA accumulation in potato in the roots and in the stems at the early infection stages compared to the leaves at later stages indicate that they are both associated to potato defenses against V. dahliae. The expression of members of the isochorismatase family in the icsh1 mutants compensate that of ICSH1 transcripts, but this compensation disappears in presence of the potato leaf extracts. This study indicates ICSH1's involvement in V. dahliae's pathogenicity and provides more insight into its alteration of the SA/JA defense signaling's networking.

Analyses of genetic diversity of bacterial blight pathogen, Xanthomonas oryzae pv. oryzae using IS1112 in Bangladesh.

Bacterial blight (BB) is caused by Xanthomonas oryzae pv. oryzae (Xoo), a most destructive disease of rice, mostly in Asia, including Bangladesh. Altogether 96 isolates of Xoo were collected from 19 rice-growing districts of Bangladesh in both the rain-fed and irrigated seasons of 2014 to assess their pathotypic and genetic variation. Pathotypic analyses were carried out on a set of 12 Near Isogenic Lines (NILs) of rice containing a single resistance gene and two check varieties IR24 and TN1 by the leaf clipping inoculation method. A total of 24 pathotypes were identified based on their virulence patterns on the NILs tested. Among these, pathotypes VII, XII and XIV, considered as major, containing a maximum number of isolates (9.38% each), are frequently distributed in seven northern to mid-eastern districts of Bangladesh. The most virulent pathotype I was recorded in the Habiganj and Brahmanbaria districts. The molecular analysis of variability among the isolates was carried out through PCR analysis using multi-locus primers Jel1 and Jel2 (based on the repetitive element IS1112 in the Xoo genome). Using the genotypic data, a dendrogram was constructed with 17 clusters along with 17 molecular haplotypes at the 65% similarity index. Cluster I was composed of 46 isolates considered as major, whereas clusters X, XI, XII and XVII were represented by a single isolate. A phenogram was constructed based on virulence to interpret the relationship between the pathotypes and the molecular haplotypes. At the 50% similarity level, among 10 clusters, cluster I, considered as major, consisted of a maximum of 10 pathotypes out of 24. In case of haplotypes, a maximum of 7 haplotypes were obtained from pathotype XII, whereas pathotypes IX, X, XV, XXII and XXIV were represented by a single haplotype. However, the present study revealed that different isolates belonging to the same pathotypes belonged to different haplotypes. Conversely, genetically similar haplotypes were also detected from different pathotypes collected from separate districts. This relationship appeared due to a high degree of DNA polymorphism among strains within many pathotypes existing in Bangladesh.

A cupin domain-containing protein with a quercetinase activity (VdQase) regulates Verticillium dahliae's pathogenicity and contributes to counteracting host defenses.

We previously identified rutin as part of potato root responses to its pathogen Verticillium dahliae. Rutin was directly toxic to the pathogen at doses greater than 160 µM, a threshold below which many V. dahliae pathogenicity-related genes were up-regulated. We identified and characterized a cupin domain-containing protein (VdQase) with a dioxygenase activity and a potential role in V. dahliae-potato interactions. The pathogenicity of VdQase knock-out mutants generated through Agrobacterium tumefasciens-mediated transformation was significantly reduced on susceptible potato cultivar Kennebec compared to wild type isolates. Fluorescence microscopy revealed a higher accumulation of flavonols in the stems of infected potatoes and a higher concentration of rutin in the leaves in response to the VdQase mutants as compared to wild type isolates. This, along with the HPLC characterization of high residual and non-utilized quercetin in presence of the knockout mutants, indicates the involvement of VdQase in the catabolism of quercetin and possibly other flavonols in planta. Quantification of Salicylic and Jasmonic Acids (SA, JA) in response to the mutants vs. wild type isolates revealed involvement of VdQase in the interference with signaling, suggesting a role in pathogenicity. It is hypothesized that the by-product of dioxygenation 2-protocatechuoylphloroglucinolcarboxylic acid, after dissociating into phloroglucinol and protocatechuoyl moieties, becomes a starting point for benzoic acid and SA, thereby interfering with the JA pathway and affecting the interaction outcome. These events may be key factors for V. dahliae in countering potato defenses and becoming notorious in the rhizosphere.

Effects of glucans and eicosapentaenoic acid on differential regulation of phenylpropanoid and mevalonic pathways during potato response to Phytophthora infestans.

The effects of Phytophthora infestans glucans, eicosapentaenoic acid (EPA) and isolates of this pathogen, on the differential expression of eight genes from the phenylpropanoid and the mevalonate (Ac-MVA) pathways were analyzed in potato by semi-quantitative RT-PCR and qRT-PCR. The application of EPA had an elicitor effect in Russet Burbank (RB) and Defender (DF) in response to inoculation with a US8 isolate of P. infestans, thereby reducing symptoms of late blight. Such effect was associated with the expression of PAL-1 and PAL-2, since the latter occurred only when EPA was followed by inoculation, whereas these genes were down-regulated in individual treatments RB + EPA, RB + US8, DF + EPA, and DF + US8. The glucan fraction did not by itself suppress phenylpropanoid genes, but its combination with the pathogen resulted in a down-regulation of PAL-1, PAL-2 and CHS. The addition of the glucan fraction to the elicitor EPA, had a negative effect (RB + EPA + GL + US8) since plants showed higher disease symptoms than the ones pretreated with water then infected with US8, and in comparison with RB + EPA + US8 and RB + GL + US8. Exclusive up-regulation of 4CL in DF + US11 and of CHS in DF + EPA + GL + US8, DF + EPA + US11, DF + GL + US11 and DF + EPA + GL + US11, where late blight lesions were not detected, could be associated with potato protection against late blight. Along with previous findings in this pathosystem, these data suggest that genetic resistance in potato against P. infestans is not the result of isolated reactions against the pathogen, but rather the combination of many factors in-line with a polygenic/horizontal resistance.

Plants versus fungi and oomycetes: pathogenesis, defense and counter-defense in the proteomics era.

Plant-fungi and plant-oomycete interactions have been studied at the proteomic level for many decades. However, it is only in the last few years, with the development of new approaches, combined with bioinformatics data mining tools, gel staining, and analytical instruments, such as 2D-PAGE/nanoflow-LC-MS/MS, that proteomic approaches thrived. They allow screening and analysis, at the sub-cellular level, of peptides and proteins resulting from plants, pathogens, and their interactions. They also highlight post-translational modifications to proteins, e.g., glycosylation, phosphorylation or cleavage. However, many challenges are encountered during in planta studies aimed at stressing details of host defenses and fungal and oomycete pathogenicity determinants during interactions. Dissecting the mechanisms of such host-pathogen systems, including pathogen counter-defenses, will ensure a step ahead towards understanding current outcomes of interactions from a co-evolutionary point of view, and eventually move a step forward in building more durable strategies for management of diseases caused by fungi and oomycetes. Unraveling intricacies of more complex proteomic interactions that involve additional microbes, i.e., PGPRs and symbiotic fungi, which strengthen plant defenses will generate valuable information on how pathosystems actually function in nature, and thereby provide clues to solving disease problems that engender major losses in crops every year.

Alteration of secondary metabolites' profiles in potato leaves in response to weakly and highly aggressive isolates of Phytophthora infestans.

Phytophthora infestans is the cause of late blight, a devastating disease in potato and tomato. Many of the mechanisms underlying P. infestans pathogenesis and defense responses in potato are still unclear. We investigated the effects of P. infestans on the changes in the accumulation of secondary metabolites in potato cultivars using whole plants. Four preformed flavonoids and one terpenoid compound produced in potato tissues were differentially affected by the P. infestans inoculation. In Russet Burbank, the accumulation of catechin and rutin was suppressed by both P. infestans isolates US-11 and US-8, while the flavanone P3 was associated with susceptibility to this pathogen. On the other hand, catechin, flavonol-glycoside P2, and an unidentified terpenoid (T1), may be involved in the defense of cultivar Defender to both tested P. infestans isolates, providing new evidence that different preformed flavonoids and terpenoids in potato may play important roles in its defense or susceptibility to P. infestans. These results add to the pool of data showing the involvement of other phenolics and terpenes in potato resistance to microbial pathogens.

Chitosan in plant protection.

Chitin and chitosan are naturally-occurring compounds that have potential in agriculture with regard to controlling plant diseases. These molecules were shown to display toxicity and inhibit fungal growth and development. They were reported to be active against viruses, bacteria and other pests. Fragments from chitin and chitosan are known to have eliciting activities leading to a variety of defense responses in host plants in response to microbial infections, including the accumulation of phytoalexins, pathogen-related (PR) proteins and proteinase inhibitors, lignin synthesis, and callose formation. Based on these and other proprieties that help strengthen host plant defenses, interest has been growing in using them in agricultural systems to reduce the negative impact of diseases on yield and quality of crops. This review recapitulates the properties and uses of chitin, chitosan, and their derivatives, and will focus on their applications and mechanisms of action during plant-pathogen interactions.