Showing papers in "Phytopathology in 2019"

High Throughput Sequencing For Plant Virus Detection and Discovery

Abstract: Over the last decade, virologists have discovered an unprecedented number of viruses using high throughput sequencing (HTS), which led to the advancement of our knowledge on the diversity of viruses in nature, particularly unraveling the virome of many agricultural crops. However, these new virus discoveries have often widened the gaps in our understanding of virus biology; the forefront of which is the actual role of a new virus in disease, if any. Yet, when used critically in etiological studies, HTS is a powerful tool to establish disease causality between the virus and its host. Conversely, with globalization, movement of plant material is increasingly more common and often a point of dispute between countries. HTS could potentially resolve these issues given its capacity to detect and discover. Although many pipelines are available for plant virus discovery, all share a common backbone. A description of the process of plant virus detection and discovery from HTS data are presented, providing a summary of the different pipelines available for scientists' utility in their research.

Current Status of Fusarium oxysporum Formae Speciales and Races.

Abstract: The Fusarium oxysporum species complex includes both plant pathogenic and nonpathogenic strains, which are commonly found in soils. F. oxysporum has received considerable attention from plant pathologists for more than a century owing to its broad host range and the economic losses it causes. The narrow host specificity of pathogenic strains has led to the concept of formae speciales, each forma specialis grouping strains with the same host range. Initially restricted to one plant species, this host range was later found to be broader for many formae speciales. In addition, races were identified in some formae speciales, generally with cultivar-level specialization. In 1981, Armstrong and Armstrong listed 79 F. oxysporum formae speciales and mentioned races in 16 of them. Since then, the known host range of F. oxysporum has considerably increased, and many new formae speciales and races have been identified. We carried out a comprehensive search of the literature to propose this review of F. oxysporum formae speciales and races. We recorded 106 well-characterized formae speciales, together with 37 insufficiently documented ones, and updated knowledge on races and host ranges. We also recorded 58 plant species/genera susceptible to F. oxysporum but for which a forma specialis has not been characterized yet. This review raises issues regarding the nomenclature and the description of F. oxysporum formae speciales and races.

Trichoderma Species: Versatile Plant Symbionts.

Abstract: Because of the need to provide food for the growing population, agricultural activity is faced with the huge challenge of counteracting the negative effects generated by adverse environmental factors and diseases caused by pathogens on crops, while avoiding environmental pollution due to the excessive use of agrochemicals. The exploitation of biological systems that naturally increase plant vigor, preparing them against biotic and abiotic stressors that also promote their growth and productivity represents a useful and viable strategy to help face these challenges. Fungi from the genus Trichoderma have been widely used in agriculture as biocontrol agents because of their mycoparasitic capacity and ability to improve plant health and protection against phytopathogens, which makes it an excellent plant symbiont. The mechanisms employed by Trichoderma include secretion of effector molecules and secondary metabolites that mediate the beneficial interaction of Trichoderma with plants, providing tolerance to biotic and abiotic stresses. Here we discuss the most recent advances in understanding the mechanisms employed by this opportunistic plant symbiont as biocontrol agent and plant growth promoter. In addition, through genome mining we approached a less explored factor that Trichoderma could be using to become successful plant symbionts, the production of phytohormones-auxins, cytokinins, abscisic acid, gibberellins, among others. This approach allowed us to detect sets of genes encoding proteins potentially involved in phytohormone biosynthesis and signaling. We discuss the implications of these findings in the physiology of the fungus and in the establishment of its interaction with plants.

Xylella fastidiosa in Olive in Apulia: Where We Stand.

Abstract: A dramatic outbreak of Xylella fastidiosa decimating olive was discovered in 2013 in Apulia, Southern Italy. This pathogen is a quarantine bacterium in the European Union (EU) and created unprecede...

Virus Detection by High-Throughput Sequencing of Small RNAs: Large-Scale Performance Testing of Sequence Analysis Strategies

Abstract: Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported but little attention has been paid thus far to their sensitivity and reliability for diagnostic purposes. Therefore, we compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large-scale performance test using 10 datasets of 21- to 24-nucleotide small RNA (sRNA) sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false-positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty in detecting viral agents when they are novel or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases, and (iv) the significant level of scientific expertise needed when interpreting pipeline results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.

Using Deep Learning for Image-Based Potato Tuber Disease Detection.

Abstract: Many plant diseases have distinct visual symptoms, which can be used to identify and classify them correctly. This article presents a potato disease classification algorithm that leverages these distinct appearances and advances in computer vision made possible by deep learning. The algorithm uses a deep convolutional neural network, training it to classify the tubers into five classes: namely, four disease classes and a healthy potato class. The database of images used in this study, containing potato tubers of different cultivars, sizes, and diseases, was acquired, classified, and labeled manually by experts. The models were trained over different train-test splits to better understand the amount of image data needed to apply deep learning for such classification tasks. The models were tested over a data set of images taken using standard low-cost RGB (red, green, and blue) sensors and were tagged by experts, demonstrating high classification accuracy. This is the first article to report the successful implementation of deep convolutional networks, popular in object identification, to the task of disease identification in potato tubers, showing the potential of deep learning techniques in agricultural tasks.

Fusarium Head Blight in Durum Wheat: Recent Status, Breeding Directions, and Future Research Prospects.

Abstract: Fusarium head blight (FHB) is a major fungal disease affecting wheat production worldwide. Since the early 1990s, FHB, caused primarily by Fusarium graminearum, has become one of the most significant diseases faced by wheat producers in Canada and the United States. The increasing FHB problem is likely due to the increased adoption of conservation tillage practices, expansion of maize production, use of susceptible wheat varieties in rotation, and climate variability. Durum wheat (Triticum turgidum sp. durum) is notorious for its extreme susceptibility to FHB and breeding for resistance is complicated because sources of FHB resistance are rare in the primary gene pool of tetraploid wheat. Losses due to this disease include yield, test weight, seed quality, food and feed quality, and when severe, market access. More importantly, it is the contamination with mycotoxins, such as deoxynivalenol, in Fusarium-infected durum kernels that causes the most serious economic as well as food and feed safety concerns. Several studies and thorough reviews have been published on germplasm development and breeding for FHB resistance and the genetics and genomics of FHB resistance in bread or common wheat (T. aestivum); however, similar reviews have not been conducted in durum wheat. Thus, the aim of this review is to summarize and discuss the recent research efforts to mitigate FHB in durum wheat, including quantitative trait locus mapping, genome-wide association studies, genomic prediction, mutagenesis and characterization of genes and pathways involved in FHB resistance. It also highlights future directions, FHB-resistant germplasm, and the potential role of morphological traits to enhance FHB resistance in durum wheat.

Mycoviruses in Biological Control: From Basic Research to Field Implementation.

Abstract: Fil: Garcia-Pedrajas, Maria D. D.. Instituto de Hortofruticultura Subtropical y Mediterrane; Espana

Progress and Obstacles in Culturing 'Candidatus Liberibacter asiaticus', the Bacterium Associated with Huanglongbing.

Abstract: In recent decades, 'Candidatus Liberibacter spp.' have emerged as a versatile group of psyllid-vectored plant pathogens and endophytes capable of infecting a wide range of economically important plant hosts. The most notable example is 'Candidatus Liberibacter asiaticus' (CLas) associated with Huanglongbing (HLB) in several major citrus-producing areas of the world. CLas is a phloem-limited α-proteobacterium that is primarily vectored and transmitted among citrus species by the Asian citrus psyllid (ACP) Diaphorina citri. HLB was first detected in North America in Florida (USA) in 2005, following introduction of the ACP to the State in 1998. HLB rapidly spread to all citrus growing regions of Florida within three years, with severe economic consequences to growers and considerable expense to taxpayers of the state and nation. Inability to establish CLas in culture (except transiently) remains a significant scientific challenge toward effective HLB management. Lack of axenic cultures has restricted functional genomic analyses, transfer of CLas to either insect or plant hosts for fulfillment of Koch's postulates, characterization of host-pathogen interactions and effective screening of antibacterial compounds. In the last decade, substantial progress has been made toward CLas culturing: (i) three reports of transient CLas cultures were published, (ii) a new species of Liberibacter was identified and axenically cultured from diseased mountain papaya (Liberibacter crescens strain BT-1), (iii) psyllid hemolymph and citrus phloem sap were biochemically characterized, (iv) CLas phages were identified and lytic genes possibly affecting CLas growth were described, and (v) genomic sequences of 15 CLas strains were made available. In addition, development of L. crescens as a surrogate host for functional analyses of CLas genes, has provided valuable insights into CLas pathogenesis and its physiological dependence on the host cell. In this review we summarize the conclusions from these important studies.

Plant Defensin Peptides have Antifungal and Antibacterial Activity Against Human and Plant Pathogens.

Abstract: Plant defensins are small, cysteine-rich antimicrobial peptides. These peptides have previously been shown to primarily inhibit the growth of fungal plant pathogens. Plant defensins have a γ-core motif, defined as GXCX3-9C, which is required for their antifungal activity. To evaluate plant defensins as a potential control for a problematic agricultural disease (alfalfa crown rot), short, chemically synthesized peptides containing γ-core motif sequences were screened for activity against numerous crown rot pathogens. These peptides showed both antifungal and, surprisingly, antibacterial activity. Core motif peptides from Medicago truncatula defensins (MtDef4 and MtDef5) displayed high activity against both plant and human bacterial pathogens in vitro. Full-length defensins had higher antimicrobial activity compared with the peptides containing their predictive γ-core motifs. These results show the future promise for controlling a wide array of economically important fungal and bacterial plant pathogens through the transgenic expression of a plant defensin. They also suggest that plant defensins may be an untapped reservoir for development of therapeutic compounds for combating human and animal pathogens.

Presence of a Sexual Population of Puccinia graminis f. sp. tritici in Georgia Provides a Hotspot for Genotypic and Phenotypic Diversity.

Abstract: Wheat stem rust, caused by Puccinia graminis f. sp. tritici, is a re-emerging disease exemplified by recent epidemics caused by new virulent races. Understanding the sources and origins of genetic ...

Trichoderma harzianum Improves Defense Against Fusarium oxysporum by Regulating ROS and RNS Metabolism, Redox Balance, and Energy Flow in Cucumber Roots

Abstract: Plant survival in the terrestrial ecosystem is influenced by both beneficial and harmful microbes. Trichoderma spp. are a group of filamentous fungi that promote plant growth and resistance to harmful microbes. Previously, we showed that the genus Trichoderma could effectively suppress Fusarium wilt in cucumber. However, the mechanisms that underlie the effects of the genus Trichoderma on plant defense have not been fully substantiated. Two essential metabolic pathways, such as the ascorbate (AsA)-glutathione (GSH) cycle and the oxidative pentose phosphate pathway (OPPP), have been shown to participate in plant tolerance to biotic stressors; nevertheless, the involvement of these pathways in Trichoderma-induced enhanced defense remains elusive. Here, we show that Trichoderma harzianum could alleviate oxidative and nitrostative stress by minimizing reactive oxygen species (ROS; hydrogen peroxide and superoxide) and reactive nitrogen species (nitric oxide [NO]) accumulation, respectively, under Fusarium oxysporum infection in cucumber roots. The genus Trichoderma enhanced antioxidant potential to counterbalance the overproduced ROS and attenuated the transcript and activity of NO synthase and nitrate reductase. The genus Trichoderma also stimulated S-nitrosylated glutathione reductase activity and reduced S-nitrosothiol and S-nitrosylated glutathione content. Furthermore, the genus Trichoderma enhanced AsA and GSH concentrations and activated their biosynthetic enzymes, γ-GCS and l-galactono-1,4-lactone dehydrogenase. Interestingly, the genus Trichoderma alleviated Fusarium-inhibited activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, enzymes involved in the OPPP. Such positive regulation of the key enzymes indicates the adequate maintenance of the AsA-GSH pathway and the OPPP, which potentially contributed to improve redox balance, energy flow, and defense response. Our study advances the current knowledge of Trichoderma-induced enhanced defense against F. oxysporum in cucumber.

Biological Control of Pierce's Disease of Grape by an Endophytic Bacterium.

Abstract: Effective preventive measures and therapies are lacking for control of Pierce's disease of grape caused by the xylem-colonizing bacterium Xylella fastidiosa responsible for serious losses in grape production. In this study we explored the potential for endophytic bacteria to alter the disease process. While most endophytic bacteria found within grape did not grow or multiply when inoculated into mature grape vines, Paraburkholderia phytofirmans strain PsJN achieved population sizes as large as 106 cells/g and moved 1 m or more within 4 weeks after inoculation into vines. While X. fastidiosa achieved large population sizes and moved extensively in grape when inoculated alone, few viable cells were recovered from plants in which it was co-inoculated with strain PsJN and the incidence of leaves exhibiting scorching symptoms typical of Pierce's disease was consistently greatly reduced from that in control plants. Suppression of disease symptoms occurred not only when strain PsJN was co-inoculated with the pathogen by puncturing stems in the same site in plants, but also when inoculated at the same time but at different sites in the plant. Large population sizes of strain PsJN could be established in both leaf lamina and petioles by topical application of cell suspensions in 0.2% of an organo-silicon surfactant conferring low surface tension, and such treatments were as effective as direct puncture inoculations of this biocontrol strain in reducing disease severity. While inoculation of strain PsJN into plants by either method at the same time as or even 4 weeks after that of the pathogen resulted in large reductions in disease severity, much less disease control was conferred by inoculation of PsJN 4 weeks prior to that of the pathogen. The expression of grapevine PR1 and ETR1 within 3 weeks of inoculation was substantially higher in plants inoculated with both X. fastidiosa and strain PsJN compared with that in plants inoculated only with the pathogen or strain PsJN, suggesting that this biological control agent reduces disease by priming expression of innate disease resistance pathways in plants that otherwise would have exhibited minimal responses to the pathogen. Strain PsJN thus appears highly efficacious for the control of Pierce's disease when used as an eradicant treatment that can be easily made even by spray application.

Nano-Magnesium Oxide: A Novel Bactericide Against Copper-Tolerant Xanthomonas perforans Causing Tomato Bacterial Spot.

Abstract: Bacterial spot caused by Xanthomonas perforans causes significant damage on tomato in Florida. Due to the presence of copper (Cu)-tolerant X. perforans strains, Cu bactericides are not effective in disease management. Hence, there is a critical need to find alternatives for Cu. Antibacterial activity of magnesium oxide (Nano-MgO), and other metal oxide nanoparticles, were evaluated against a Cu-tolerant and -sensitive X. perforans strain. In vitro experiments demonstrated high antibacterial activity of Nano-MgO against both strains compared with the commercial Cu. The minimum inhibitory concentration of Nano-MgO is 25 µg/ml and the minimum bactericidal concentration is 100 µg/ml against a Cu-tolerant X. perforans strain after 4 h of exposure. Structural changes in the bacterial membrane following exposure to Nano-MgO treatments compared with the controls were observed using transmission electron microscopy. In two greenhouse experiments with a Cu-tolerant strain, bacterial spot severity was significantly reduced by Nano-MgO at 200 µg/ml compared with Cu-ethylene bis-dithiocarbamate (grower standard), and the untreated control (P = 0.05). In three field experiments, Nano-MgO at 200 µg/ml significantly reduced disease severity with no negative impact on yield compared with the untreated control. Inductively coupled plasma mass spectrometric analysis of the fruit confirmed that Nano-MgO application did not lead to the accumulation of Mg, Cu, Ca, K, Mn, P, and S. This study is the first to demonstrate the potential of Nano-MgO against bacterial spot of tomato.

Chocolate Under Threat from Old and New Cacao Diseases.

Abstract: Theobroma cacao, the source of chocolate, is affected by destructive diseases wherever it is grown. Some diseases are endemic; however, as cacao was disseminated from the Amazon rain forest to new cultivation sites it encountered new pathogens. Two well-established diseases cause the greatest losses: black pod rot, caused by several species of Phytophthora, and witches' broom of cacao, caused by Moniliophthora perniciosa. Phytophthora megakarya causes the severest damage in the main cacao producing countries in West Africa, while P. palmivora causes significant losses globally. M. perniciosa is related to a sister basidiomycete species, M. roreri which causes frosty pod rot. These Moniliophthora species only occur in South and Central America, where they have significantly limited production since the beginnings of cacao cultivation. The basidiomycete Ceratobasidium theobromae causing vascular-streak dieback occurs only in South-East Asia and remains poorly understood. Cacao swollen shoot disease caused by Cacao swollen shoot virus is rapidly spreading in West Africa. This review presents contemporary research on the biology, taxonomy and genomics of what are often new-encounter pathogens, as well as the management of the diseases they cause.

Proteomic and Metabolomic Analyses of Xylella fastidiosa OMV-Enriched Fractions Reveal Association with Virulence Factors and Signaling Molecules of the DSF Family

Abstract: Xylella fastidiosa releases outer membrane vesicles (OMVs) known to play a role in the systemic dissemination of this pathogen. OMVs inhibit bacterial attachment to xylem wall and traffic lipases/esterases that act on the degradation of plant cell wall. Here, we extended the characterization of X. fastidiosa OMVs by identifying proteins and metabolites potentially associated with OMVs produced by Temecula1, a Pierce's disease strain, and by 9a5c and Fb7, two citrus variegated chlorosis strains. These results strengthen that one of the OMVs multiple functions is to carry determinants of virulence, such as lipases/esterases, adhesins, proteases, porins, and a pectin lyase-like protein. For the first time, we show that the two citrus variegated chlorosis strains produce X. fastidiosa diffusible signaling factor 2 (DSF2) and citrus variegated chlorosis-DSF (likewise, Temecula1) and most importantly, that these compounds of the DSF (X. fastidiosa DSF) family are associated with OMV-enriched fractions. Altogether, our findings widen the potential functions of X. fastidiosa OMVs in intercellular signaling and host-pathogen interactions.

Developing Citrus Huanglongbing (HLB) Management Strategies Based on the Severity of Symptoms in HLB-Endemic Citrus-Producing Regions

Abstract: Citrus Huanglongbing (HLB), also known as greening, is a destructive disease caused by the fastidious, phloem-colonizing bacteria Candidatus Liberibacter spp.; 'Ca. Liberibacter asiaticus' (Las) is the most prevalent of the species causing HLB. The Asian citrus psyllid (ACP, Diaphorina citri) transmits Las. HLB is threatening citrus production worldwide, and there is no cure for infected trees. Management strategies targeting diseased trees at different stages of colonization by Las are needed for sustainable citrus production in HLB-endemic regions. We evaluated the effect of the combinations of plant defense elicitors, nitrogen (N) fertilizer, and compost on mildly diseased trees. We tested thermotherapy on severely diseased trees and assessed tree protectors to prevent feeding by ACP, thus preventing Las from being transmitted to new plantings that replaced HLB-moribund trees. After four applications over two consecutive growing seasons we found that the combination of compost, urea, and plant defense elicitors β-aminobutyric acid, plus ascorbic acid and potassium phosphite with or without salicylic acid, slowed down the progression of HLB and reduced disease severity by approximately 18%, compared with the untreated control. Our data showed no decline in fruit yield, indeed treatment resulted in a higher yield compared with the untreated control. Thermotherapy treatment (55°C for 2 min) exhibited a suppressive effect on growth of Las and progress of HLB in severely diseased trees for 2 to 3 months after treatment. The tree protectors prevented feeding by ACP, and therefore young replant trees remained healthy and free from infection by Las over the 2-year duration of the experiment. Taken together, these results may contribute to a basis for developing a targeted approach to control HLB based on stage of host colonization, application of plant defense elicitors, N fertilizer, compost, thermotherapy, and tree protectors. There is potential to implement these strategies in conjunction with other disease control measures to contribute to sustainable citrus production in HLB-endemic regions.

Short-Term Effects of Sublethal Doses of Nematicides on Meloidogyne incognita

Abstract: Meloidogyne species are one of the most important groups of plant-parasitic nematodes globally because of their ability to damage most cultivated plants. Although they are widespread and economically important, there are limited control measures to combat these nematodes. New nonfumigant nematicides have been discovered that have the potential to be widely utilized for the management of plant-parasitic nematodes. Because of the longer half-lives in soil and lower toxicity of new nematicides compared with traditional fumigant and nonfumigant nematicides, understanding how nematodes respond to sublethal doses of nematicides is imperative to understanding whether nematicide resistance has the potential to develop. Characterizing responses of nematodes to sublethal doses will provide the foundation for future work, such as gene expression studies. In this study, the nematicides oxamyl (Vydate), fluazaindolizine (Salibro), fluensulfone (Nimitz), and fluopyram (Velum), were evaluated to understand how sublethal doses affect the fecundity and mobility of Meloidogyne incognita second-stage juveniles (J2). Using a microwell assay system, dose-response curves for each nematicide were established for M. incognita J2. Fluopyram was the most toxic nematicide, with effective doses up to 230 times lower than that of other nematicides. The other nematicides had predicted ED50 values (effective doses that resulted in 50% of the population becoming inactive) of 89.4, 131.7, and 180.6 ppm for oxamyl, fluensulfone, and fluazaindolizine, respectively. The 24-h ED50 of each nematicide was then used in both motility and infectivity assays. The motility and activity of M. incognita J2 exposed to ED50 doses of fluazaindolizine and fluensulfone was significantly reduced, with nematodes initially being motile but eventually becoming inactive. However, the motility of M. incognita J2 exposed to ED50 doses of fluopyram and oxamyl was not different from a water control. In a pot assay, M. incognita J2 exposed to ED50 doses of fluazaindolizine, oxamyl, and fluensulfone were unable to reproduce on tomato, with reproduction factors (RF = final population density/initial population density) of 0 to 0.03. Fluopyram did not reduce reproduction of M. incognita, with a mean RF of 38.7 ± 4.5, which was similar to the RF of 46.3 ± 4.6 for the water control. This study is the first comprehensive evaluation of M. incognita activity, motility, and fecundity after exposure to the traditional nematicide, oxamyl, as well as three new nematicides, fluazaindolizine, fluopyram, and fluensulfone.

The in Planta Effective Concentration of Oxytetracycline Against 'Candidatus Liberibacter asiaticus' for Suppression of Citrus Huanglongbing.

Abstract: Huanglongbing (HLB) or greening currently is the most devastating citrus disease worldwide. The fastidious phloem-colonizing bacterium 'Candidatus Liberibacter asiaticus' (CLas) is the causal agent of citrus HLB in Florida. Bactericides containing the active ingredient oxytetracycline (OTC) have been used in foliar spray to control citrus HLB in Florida since 2016. However, the minimum concentration of OTC required to suppress CLas in planta remains unknown. We developed a new method for evaluating the effects of OTC treatment on CLas titers in infected plants and determined the relationship between OTC residue levels and control levels achieved for CLas using mathematical modeling in greenhouse and field experiments. In both greenhouse and field, OTC spray did not reduce the titers of CLas, and it produced undetectable or mild levels of OTC residue in leaves within 7 days post-application (DPA). In greenhouse, OTC injection at 0.05 g per tree decreased CLas titers to an undetectable level (cycle threshold value ≥ 36.0) from 7 to 30 DPA and produced a residue level of OTC at 0.68 to 0.73 µg/g of fresh tissue over this period. In the field, OTC injection at 0.50 g per tree resulted in the decline of CLas titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27 to 0.33 µg/g of fresh tissue. In both trials, a first-order compart model of OTC residue dynamics in leaves of trunk-injected trees was specified for estimating the retention of effective concentrations. Furthermore, nonlinear modeling revealed significant positive correlations between OTC residue levels in leaves and the control levels for CLas achieved. The results suggested that the minimum concentrations of OTC required to suppress CLas populations in planta to below the detection limit are 0.68 and 0.86 µg/g and that the minimum concentrations of OTC required for initial inhibition of CLas growth in planta are ∼0.17 and ∼0.215 µg/g in leaf tissues under greenhouse and field conditions, respectively. This finding highlights that a minimum concentration of OTC should be guaranteed to be delivered to target CLas in infected plants for effective control of citrus HLB.

miR156/SPL9 Regulates Reactive Oxygen Species Accumulation and Immune Response in Arabidopsis thaliana

Abstract: The functions of microRNA156 (miR156) and its targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor genes in plant development have been widely investigated. However, the role of the miR156/SPLs regulatory network in plant immune systems remains obscure. Here, we found that the accumulation of reactive oxygen species (ROS) and the transcripts of basal salicylic acid (SA) signaling pathway genes were lower in Arabidopsis Pro35S:MIR156 seedlings (miR156 overexpression mutants) but higher in Pro35S:MIM156 (miR156 repression mutants) and ProSPL9:rSPL9 (SPL9 overexpression mutants) seedlings compared with wild-type Col-0 plants (WT). As a result, Pro35S:MIR156 mutants induced greater susceptibility to Pseudomonas syringae pv. tomato DC3000 following syringe infiltration than WT, while Pro35S:MIM156 and ProSPL9:rSPL9 mutants showed enhanced resistance. In addition, foliar H2O2 application resulted in activation of SA-mediated defense response and ablation of miR156-induced susceptibility to P. syringae pv. tomato DC3000 infection. Collectively, our results provide new insights into the function of the miR156/SPL network in Arabidopsis immune response by regulating ROS accumulation and activating the SA signaling pathway.

A Bacterial Endophyte from Apoplast Fluids Protects Canola Plants from Different Phytopathogens via Antibiosis and Induction of Host Resistance.

Abstract: Endophytic bacteria colonize inner plant tissues and thrive at the apoplast, which is considered its main reservoir. Because this niche is the place where the main molecular events take place between beneficial and pathogenic microorganisms, the aim of this work was to characterize culturable endophytic bacteria from apoplastic fluids obtained from field-grown canola leaves and analyze their potential for biological control of diseases caused by Xanthomonas campestris, Sclerotinia sclerotiorum, and Leptosphaeria maculans. Dual-culture analysis indicated that three isolates (Apo8, Apo11, and Apo12) were able to inhibit the growth of all three phytopathogens. Sequencing of the 16S ribosomal RNA and rpoD genes of these isolates revealed that they are closely related to Pseudomonas viridiflava. One of the isolates, Apo11, was able to diminish the propagation of X. campestris in whole-plant assays. At the same time, Apo11 inoculation reduced the necrotic lesions provoked by S. sclerotiorum on canola leaves. This protective effect might be due to the induction of resistance in the host mediated by salicylic and jasmonic acid signaling pathways or the production of compounds with antimicrobial activity. At the same time, Apo11 inoculation promoted canola plant growth. Thus, the isolate characterized in this work has several desirable characteristics, which make it a potential candidate for the formulation of biotechnological products to control plant diseases or promote plant growth.

Characterization of Antagonistic Bacillus methylotrophicus Isolated From Rhizosphere and Its Biocontrol Effects on Maize Stalk Rot.

Abstract: Stalk rot is one of the most serious and widespread diseases in maize, and effective control measures are currently lacking. Therefore, this study aimed to develop a new biological agent to manage this disease. An antagonistic bacterial strain, TA-1, was isolated from rhizosphere soil and identified as Bacillus methylotrophicus based on morphological and biochemical characterization and 16S ribosomal RNA and gyrB gene sequence analyses. TA-1 exhibited a strong antifungal effect on the growth of Fusarium graminearum mycelium, with 86.3% inhibition at a concentration of 108 CFU per ml. Transmission electron microscopy showed that TA-1 could disrupt the cellular structure of the fungus, induce necrosis, and degrade the cell wall. Greenhouse and field trials were performed to evaluate the biocontrol efficacy of TA-1 on maize stalk rot, and the results of greenhouse experiment revealed that the bacterium significantly reduced disease incidence and disease index. Seeds treated with a 108 CFU ml-1 cell suspension had the highest disease suppression at 86.8%. Results of field trials show that seed bacterization with TA-1 could not only reduce maize stalk rot incidence but also increase maize height, stem diameter, and grain yield. The lipopeptide antibiotics were isolated from the culture supernatants of TA-1 and identified as surfactins and iturins. Consequently, B. methylotrophicus TA-1 is a potential biocontrol agent against maize stalk rot.

Necrotrophic Exploitation and Subversion of Plant Defense: A Lifestyle or Just a Phase, and Implications in Breeding Resistance.

Abstract: Breeding disease-resistant plants is a critical, environmentally friendly component of any strategy to sustainably feed and clothe the 9.8 billion people expected to live on Earth by 2050. Here, I review current literature detailing plant defense responses as they relate to diverse biological outcomes; disease resistance, susceptibility, and establishment of mutualistic plant-microbial relationships. Of particular interest is the degree to which these outcomes are a function of plant-associated microorganisms' lifestyles; biotrophic, hemibiotrophic, necrotrophic, or mutualistic. For the sake of brevity, necrotrophic pathogens and the necrotrophic phase of pathogenicity are emphasized in this review, with special attention given to the host-specific pathogens that exploit defense. Defense responses related to generalist necrotrophs and mutualists are discussed in the context of excellent reviews by others. In addition, host evolutionary trade-offs of disease resistance with other desirable traits are considered in the context of breeding for durable disease resistance.

The Involvement of Jasmonic Acid, Ethylene, and Salicylic Acid in the Signaling Pathway of Clonostachys rosea-Induced Resistance to Gray Mold Disease in Tomato.

Abstract: Tomato gray mold disease caused by Botrytis cinerea is a serious disease that threatens tomato production around the world. Clonostachys rosea has been used successfully as a biocontrol agent against divergent plant pathogens, including B. cinerea. To understand the signal transduction pathway of C. rosea-induced resistance to tomato gray mold disease, the effects of C. rosea on gray mold tomato leaves along with changes in the activities of three defense enzymes (phenylalanine ammonialyase [PAL], polyphenol oxidase [PPO], and catalase [CAT]), second messengers (nitric oxide [NO], hydrogen peroxide [H2O2], and superoxide anion radical [O2-]), and stress-related genes (mitogen-activated protein kinase [MAPK], WRKY, Lexyl2, and atpA) in four different hormone-deficient (jasmonic acid [JA], ethylene [ET], salicylic acid [SA], and gibberellin) tomato mutants were investigated. The results revealed that C. rosea significantly inhibited the growth of mycelia and spore germination of B. cinerea. Furthermore, it reduced the incidence of gray mold disease, induced higher levels of PAL and PPO, and induced lower levels of CAT activities in tomato leaves. Moreover, it also increased NO, H2O2, and O2- levels and the gene expression levels of WRKY, MAPK, atpA, and Lexyl2. The incidence of gray mold disease in four hormone-deficient mutants was higher than that in the corresponding wild-type tomato plants. Among all of these hormone-deficient tomato mutants, JA had the most significant effect in regulating the different signal molecules. Additional study suggested that JA upregulated the expression of Lexyl2, MAPK, and WRKY but downregulated atpA. Furthermore, JA also enhanced the activity of PAL, PPO, and CAT and the production of NO and H2O2. SA downregulated CAT and PAL, whereas ET upregulated PAL but downregulated CAT. This study is of significance in understanding the regulatory pathways and biocontrol mechanism of C. rosea against B. cinerea.

Identification and Characterization of Plasmodiophora brassicae Primary Infection Effector Candidates that Suppress or Induce Cell Death in Host and Nonhost Plants.

Abstract: Clubroot caused by Plasmodiophora brassicaeis one of the most important diseases in cruciferous crops. The recognition of P. brassicae by host plants is thought to occur at the primary infection stage, but the underlying mechanism remains unclear. Secretory proteins as effector candidates play critical roles in the recognition of pathogens and the interactions between pathogens and hosts. In this study, 33 P. brassicae secretory proteins expressed during primary infection were identified through transcriptome, secretory protein prediction, and yeast signal sequence trap analyses. Furthermore, the proteins that could suppress or induce cell death were screened through an Agrobacterium-mediated plant virus transient expression system and a protoplast transient expression system. Two secretory proteins, PBCN_002550 and PBCN_005499, were found to be capable of inducing cell death associated with H2O2 accumulation and electrolyte leakage in Nicotiana benthamiana. Moreover, PBCN_002550 could also induce cell death in Chinese cabbage. In addition, 24 of the remaining 31 tested secretory proteins could suppress mouse Bcl-2-associated X protein-induced cell death, and 28 proteins could suppress PBCN_002550-induced cell death.

Identifying Lookouts for Epidemio-Surveillance: Application to the Emergence of Xylella fastidiosa in France.

Abstract: Recent detections of Xylella fastidiosa in Corsica Island, France, has raised concerns on its possible spread to mainland France and the rest of the Mediterranean Basin. Early detection of infected plants is paramount to prevent the spread of the bacteria, but little is known about this pathosystem in European environments, hence standard surveillance strategies may be ineffective. We present a new methodological approach for the design of risk-based surveillance strategies, adapted to the emerging risk caused by X. fastidiosa. Our proposal is based on a combination of machine learning techniques and network analysis that aims at understanding the main abiotic drivers of the infection, produce risk maps and identify lookouts for the design of future surveillance plans. The identified drivers coincide with known results in laboratory studies about the correlation between environmental variables, such as water stress and temperature, and the presence of the bacterium in plants. Furthermore, the produced risk maps overlap nicely with detected foci of infection, while they also highlight other susceptible regions where X. fastidiosa has not been found yet. We conclude the paper presenting a list of recommended regions for a risk-based surveillance campaign based on the predicted spread and probability of early detection of the disease.

Metabolome and microbiome signatures in the roots of citrus affected by Huanglongbing.

Abstract: Huanglongbing (HLB) is a severe, incurable citrus disease caused by the bacterium 'Candidatus Liberibacter asiaticus' (CLas). Although citrus leaves serve as the site of initial infection, CLas is known to migrate to and colonize the root system; however, little is known about the impact of CLas infection on root metabolism and resident microbial communities. Scions of 'Lisbon' lemon and 'Washington Navel' orange grafted onto 'Carrizo' rootstock were grafted with either CLas-infected citrus budwood or uninfected budwood. Roots were obtained from trees 46 weeks after grafting and analyzed via 1H nuclear magnetic resonance spectroscopy to identify water-soluble root metabolites and high-throughput sequencing of 16S rRNA and ITS gene amplicons to determine the relative abundance of bacterial and fungal taxa in the root rhizosphere and endosphere. In both citrus varieties, 27 metabolites were identified, of which several were significantly different between CLas(+) and control plants. CLas infection also appeared to alter the microbial community structure near and inside the roots of citrus plants. Nonmetric multidimensional scaling (NMDS) and a principal coordinate analysis (PCoA) revealed distinct metabolite and microbial profiles, demonstrating that CLas impacts the root metabolome and microbiome in a manner that is variety-specific.

Recombinase Polymerase Amplification Assay for Field Detection of Tomato Bacterial Spot Pathogens

Abstract: Bacterial spot of tomato is caused by Xanthomonas gardneri, X. euvesicatoria, X. perforans, and X. vesicatoria. Current diagnostic methods for the pathogens are not in-field assays. Recombinase polymerase amplification (RPA) is ideal for in-field detection assays, because it is an isothermal technique that is rapid and more tolerant to inhibitors compared with polymerase chain reaction. Hence, novel RPA probes and primers were designed to amplify regions of the hrcN gene of X. gardneri, X. euvesicatoria, and X. perforans. The X. gardneri RPA is specific to X. gardneri with a detection limit of 106 CFU/ml and detected X. gardneri in lesions from naturally (n = 6) or artificially (n = 18) infected plants. The X. euvesicatoria RPA detects both X. euvesicatoria and X. perforans with a detection limit of 106 CFU/ml and detected both pathogens in plants artificially infected (n = 36) or naturally infected (n = 85) with either X. euvesicatoria or X. perforans. The X. perforans RPA is specific to X. perforans with a detection limit of 107 CFU/ml. Although the X. perforans RPA assay was unable to detect X. perforans from lesions, the X. euvesicatoria RPA was successfully used in field to detect X. perforans from symptomatic field samples (n = 31). The X. perforans RPA was then used to confirm the pathogen in the laboratory. The X. euvesicatoria and X. gardneri RPA is promising for rapid, real-time in-field detection of bacterial spot and one of the first developed among plant pathogenic bacteria.

Time to fight: molecular mechanisms of age-related resistance.

Abstract: Plant age is a crucial factor in determining the outcome of a host-pathogen interaction. In successive developmental stages throughout their life cycles, plants face dynamic changes in biotic and abiotic conditions that create distinct ecological niches for host-pathogen interactions. As an adaptive strategy, plants have evolved intrinsic regulatory networks that integrate developmental signals with those from pathogen perception and defense activation. As a result, amplitude and timing of defense responses are optimized, so as to balance the cost and benefit of immunity activation. A general term "age-related resistance" refers to a gain of disease resistance against a certain pathogen when plants reach a relatively mature stage. Age-related resistance is a common observation on fruits, vegetables, and row crops for their resistance against viruses, bacteria, fungi, oomycetes pathogens, and insects. This review focuses on the recent advances in understanding the molecular mechanisms of how plants coordinate developmental timing and immune response.

Prophage Diversity of 'Candidatus Liberibacter asiaticus' Strains in California.

Abstract: Huanglongbing (HLB) is a highly destructive citrus disease and is associated with a nonculturable bacterium, 'Candidatus Liberibacter asiaticus'. 'Ca. L. asiaticus' in the United States was first found in Florida in 2005 and is now endemic there. In California, 'Ca. L. asiaticus' was first detected in Hacienda Heights in Los Angeles County in 2012 and has now been detected in multiple urban locations in southern California. Knowledge of 'Ca. L. asiaticus' strain diversity in California is important for HLB management. In this study, genomic diversity among 10 'Ca. L. asiaticus' strains from six California locations were analyzed using a next-generation sequencing (NGS) (Illumina MiSeq and HiSeq) approach. Draft genome sequences of 'Ca. L. asiaticus' strains were assembled. Sequences of the 16S ribosomal RNA gene and nrdB confirmed 'Ca. L. asiaticus' identity. Prophages were detected in all 'Ca. L. asiaticus' strains. The California 'Ca. L. asiaticus' strains formed four prophage typing groups (PTGs): PTG1, with type 1 prophage only (strains from Anaheim, San Gabriel, and Riverside); PTG2, with type 2 prophage only (strains from Hacienda Heights); PTG1-3, with both type 1 and 3 prophages (a strain from Cerritos); and PTG1-2, with both type 1 and type 2 prophages (a strain from La Habra). Analyses of the terL sequence showed that all California 'Ca. L. asiaticus' strains were not introduced from Florida but likely from locations in Asia. Miniature inverted-repeat transposable elements were found in all 'Ca. L. asiaticus' strains, yet, a jumping-out event was detected in the 'Ca. L. asiaticus' strain from Cerritos. Altogether, this study demonstrated that the NGS approach focusing on prophage variation was sensitive and effective in revealing diversity of 'Ca. L. asiaticus' strains in California.