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Correction FRONTIERS IN MICROBIOLOGY Volume: 12 Article Number: 693341 DOI: 10.3389/fmicb.2021.693341 Published: MAY 20 2021 The soil-borne plant pathogens cause serious yield losses and are difficult to control. In suppressive soils, disease incidence remains low regardless of the presence of the pathogen, the host plant, and favorable environmental conditions. The potential to improve natural soil disease suppressiveness through agricultural management practices would enable sustainable and resilient crop production systems. Our aim was to study the impact of autumn tillage methods and crop sequence on the soil carbon status, fungistasis and yield in boreal climate. The disease suppression was improved by the long-term reduced and no tillage management practices with and without crop rotation. Compared to the conventional plowing, the non-inversion tillage systems were shown to change the vertical distribution of soil carbon fractions and the amount of microbial biomass by concentrating them on the soil surface. Crop sequence and the choice of tillage method had a combined effect on soil organic carbon (SOC) sequestration. The improved general disease suppression had a positive correlation with the labile carbon status and microbial biomass. From the most common Fusarium species, the predominantly saprophytic F. avenaceum was more abundant under non-inversion practice, whereas the opposite was true for the pathogenic ones. Our findings furthermore demonstrated the correlation of the soil fungistasis laboratory assay results and the prevalence of the pathogenic test fungus Fusarium culmorum on the crop cereals in the field. Our results indicate that optimized management strategies have potential to improve microbial related soil fungistasis in boreal climate.

Fusarium root rot is a major cryptogamic disease in olive trees caused by the soil-borne fungus Fusarium solani. Controlling this disease requires the extensive use of chemicals. However, using BCAs such as some Trichoderma strains may be an opportune alternative to fungicides in protecting olive plantations. A new isolate (Fso14) was isolated from young olive trees showing severe dieback symptoms. The objective of this work was to analyze the biocontrol behavior of a Tunisian strain of T. harzianum (Ths97) on olive trees against Fso14 by assessing both mycoparasitic activity (in planta and in vitro) and ability to locally modulate different gene-related defenses of the plant. Ths97 was found to inhibit Fso14 growth in vitro. Optical microscopic analysis at the confrontation zone between hyphae showed that Ths97 grew alongside Fso14 with numerous contact points suggesting parasitic activity. On olive trees, Ths97 developed a strong protective role against root infestation by Fso14, whether inoculated before or after the pathogenic agent. When inoculated alone, Fso14 and Ths97 did not modulate (or only slightly with inhibitions or inductions, respectively) the expression of genes involved in plant immunity (oxidative stress, phenylpropanoid pathway, PR-proteins and JA/Et-SA hormonal status). However, when Ths97 was inoculated in combination with Fso14, several defense-related genes were highly up-regulated, indicating probable primed-plant events. These promising results provided valuable information on using Ths97 as a beneficial agent to control fusarium root rot disease caused by F. solani in olive trees.

Crude extracts of in vitro and in vivo cultures of two strains of Photorhabdus l. sonorensis (Enterobacteriaceae) were analyzed by TLC, HPLC-UV and LC-MS. Nine unique compounds with mass/charge ratios (m/z) ranging from 331.3 to 713.5 were found in MS analyses. Bioactivity of extracts was assessed on a selection of plant pathogens/pests and non-target species. Caborca strain extracts showed the highest activity against Helicoverpa zea (Lepidoptera: Noctuidae) neonates at all concentrations tested. Mortality ranged from 11% (at 10 μg/ml) to 37% (at 40 μg/ml). Strain CH35 extracts showed the highest nematicidal activity on Meloidogyne incognita (Tylenchida: Meloidogynidae) at 40 μg/ml. Low to no nematicidal activity was observed against the non-target species Steinernema carpocapsae (Rhabditida: Steinernematidae) and Caenorhabditis elegans (Rhabditida: Rhabditidae). Caborca extracts exhibited a strong antibiotic effect on Pseudomonas syringae (Pseudomonadales: Pseudomonadacedae) at 40 μg/ml, while both Caborca and CH35 extracts inhibited the growth of Bacillus subitillis (Bacillales: Bacillaceae) at 40 μg/ml. All extracts strongly inhibited the growth of the fungus Fusarium oxysporum (Hypocreales: Nectriceae) but not that of Alternaria alternata (Pleosporales: Pleosporaceae). Contrastingly, a moderate to high inhibitory effect was denoted on the non-target biocontrol fungus Beauveria bassiana (Hypocreales: Clavivipitaceae).

During plant cell invasion, the oomycete Phytophthora infestans remains enveloped by host-derived membranes whose functional properties are poorly understood. P. infestans secretes a myriad of effector proteins through these interfaces for plant colonization. Recently we showed that the effector protein PexRD54 reprograms host-selective autophagy by antagonising antimicrobial-autophagy receptor Joka2/NBR1 for ATG8CL binding (Dagdas et al., 2016). Here, we show that during infection, ATG8CL/Joka2 labelled defense-related autophagosomes are diverted toward the perimicrobial host membrane to restrict pathogen growth. PexRD54 also localizes to autophagosomes across the perimicrobial membrane, consistent with the view that the pathogen remodels host-microbe interface by co-opting the host autophagy machinery. Furthermore, we show that the host-pathogen interface is a hotspot for autophagosome biogenesis. Notably, overexpression of the early autophagosome biogenesis protein ATG9 enhances plant immunity. Our results implicate selective autophagy in polarized immune responses of plants and point to more complex functions for autophagy than the widely known degradative roles. Fil: Dagdas, Yasin F.. Norwich Research Park; Reino Unido. Vienna Biocenter; Austria Fil: Pandey, Pooja. Imperial College London; Reino Unido Fil: Tumtas, Yasin. Imperial College London; Reino Unido Fil: Sanguankiattichai, Nattapong. Imperial College London; Reino Unido Fil: Belhaj, Khaoula. Norwich Research Park; Reino Unido Fil: Duggan, Cian. Imperial College London; Reino Unido Fil: Leary, Alexandre Y.. Imperial College London; Reino Unido Fil: Segretin, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Contreras, Mauricio P.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Imperial College London; Reino Unido Fil: Savage, Zachary. Imperial College London; Reino Unido Fil: Khandare, Virendrasinh S.. Imperial College London; Reino Unido Fil: Kamoun, Sophien. Norwich Research Park; Reino Unido Fil: Bozkurt, Osman Tolga. Imperial College London; Reino Unido

Powdery mildews are biotrophic pathogenic fungi infecting a number of economically important plants. The grass powdery mildew,Blumeria graminis,has become a model organism to study host specialization of obligate biotrophic fungal pathogens. We resolved the large-scale genomic architecture ofB. graminis forma specialis hordei (Bgh)to explore the potential influence of its genome organization on the co-evolutionary process with its host plant, barley(Hordeum vulgare).The near-chromosome level assemblies of theBghreference isolate DH14 and one of the most diversified isolates, RACE1, enabled a comparative analysis of these haploid genomes, which are highly enriched with transposable elements (TEs). We found largely retained genome synteny and gene repertoires, yet detected copy number variation (CNV) of secretion signal peptide-containing protein-coding genes (SPs) and locally disrupted synteny blocks. Genes coding for sequence-related SPs are often locally clustered, but neither theSPclusters nor TEs are enriched in specific genomic regions. Extended comparative analysis with different host-specificB. graminis formae specialesrevealed the existence of a core suite ofSPs,but also isolate-specificSPsets as well as congruence ofSPCNV and phylogenetic relationship. We further detected evidence for a recent, lineage-specific expansion of TEs in theBghgenome. The characteristics of theBghgenome (largely retained synteny, CNV ofSPgenes, recently proliferated TEs and a lack of compartmentalization) are consistent with a “one-speed” genome that differs in its architecture and (co-)evolutionary pattern from the “two-speed” genomes reported for several other filamentous phytopathogens.

Accelerating international trade and climate change make pathogen spread an increasing concern. Hymenoscyphus fraxineus, the causal agent of ash dieback, is a fungal pathogen that has been moving across continents and hosts from Asian to European ash. Most European common ash trees (Fraxinus excelsior) are highly susceptible to H. fraxineus, although a minority (~5%) have partial resistance to dieback. Here, we assemble and annotate a H. fraxineus draft genome, which approaches chromosome scale. Pathogen genetic diversity across Europe and in Japan, reveals a strong bottleneck in Europe, though a signal of adaptive diversity remains in key host interaction genes. We find that the European population was founded by two divergent haploid individuals. Divergence between these haplotypes represents the ancestral polymorphism within a large source population. Subsequent introduction from this source would greatly increase adaptive potential of the pathogen. Thus, further introgression of H. fraxineus into Europe represents a potential threat and Europe-wide biological security measures are needed to manage this disease.

Climate changes, exemplified by increased temperatures and CO2 concentration, pose a global threat to forest health. Of particular concern are pests and pathogens, with a warming climate altering their distributions and evolutionary capacity, while impairing the ability of some plants to respond to infections. Progress in understanding and mitigating such effects is currently hindered by a lack of empirical research. Norway spruce (Picea abies) is one of the most economically important tree species in northern Europe, and is considered highly vulnerable to changes in climate. It is commonly infected by the fungus Endoconidiophora polonica, and we hypothesized that damage caused to trees will increase under future climate change predictions. To test this hypothesis an in vivo greenhouse experiment was conducted to evaluate the effects of a changed growing environment on E. polonica infected Norway spruce seedlings, comparing ambient conditions to predicted temperatures and CO2 levels in Finland for the years 2030 and 2100. In total, 450 seedlings were randomized amongst the three treatments, with 25 seedlings from each allocated to inoculation with one of five different fungal strains or mock-inoculation. Seedlings were monitored throughout the thermal growing season for mortality, and lesion length and depth indices were measured at the experiment conclusion. Disease severity (mortality and lesions) was consistently greater in fungal-inoculated than mock-inoculated seedlings. However, substantial differences were observed among fungal strains in response to climate scenarios. For example, although overall seedling mortality was highest under the most distant (and severe) climate change expectations, of the two fungal strains with the highest mortality counts (referred to as F4 and F5), one produced greater mortality under the 2030 and 2100 scenarios than ambient conditions, whereas climate scenario had no effect on the other. This study contributes to a limited body of empirical research on the effects of projected climate changes on forestry pathosystems, and is the first to investigate interactions between Norway spruce and E. polonica. The results indicate the potential for future climate changes to alter the impact of forest pathogens with implications for productivity, while highlighting the need for a strain-specific level of understanding of the disease agents.

Maize lethal necrosis (MLN) has emerged as a serious threat to food security in sub-Saharan Africa. MLN is caused by coinfection with two viruses, Maize chlorotic mottle virus and a potyvirus, often Sugarcane mosaic virus. To better understand the dynamics of MLN and to provide insight into disease management, we modeled the spread of the viruses causing MLN within and between growing seasons. The model allows for transmission via vectors, soil, and seed, as well as exogenous sources of infection. Following model parameterization, we predict how management affects disease prevalence and crop performance over multiple seasons. Resource-rich farmers with large holdings can achieve good control by combining clean seed and insect control. However, crop rotation is often required to effect full control. Resource-poor farmers with smaller holdings must rely on rotation and roguing, and achieve more limited control. For both types of farmer, unless management is synchronized over large areas, exogenous sources of infection can thwart control. As well as providing practical guidance, our modeling framework is potentially informative for other cropping systems in which coinfection has devastating effects. Our work also emphasizes how mathematical modeling can inform management of an emerging disease even when epidemiological information remains scanty. [Formula: see text] Copyright © 2017 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

While governments and individuals strive to maintain the availability of high-quality water resources, many factors can “change the landscape” of water availability and quality, including drought, climate change, saltwater intrusion, aquifer depletion, population increases, and policy changes. Specialty crop producers, including nursery and greenhouse container operations, rely heavily on available high-quality water from surface and groundwater sources for crop production. Ideally, these growers should focus on increasing water application efficiency through proper construction and maintenance of irrigation systems, and timing of irrigation to minimize water and sediment runoff, which serve as the transport mechanism for agrichemical inputs and pathogens. Rainfall and irrigation runoff from specialty crop operations can contribute to impairment of groundwater and surface water resources both on-farm and into the surrounding environment. This review focuses on multiple facets of water use, reuse, and runoff in nursery and greenhouse production including current and future regulations, typical water contaminants in production runoff and available remediation technologies, and minimizing water loss and runoff (both on-site and off-site). Water filtration and treatment for the removal of sediment, pathogens, and agrichemicals are discussed, highlighting not only existing understanding but also knowledge gaps. Container-grown crop producers can either adopt research-based best management practices proactively to minimize the economic and environmental risk of limited access to high-quality water, be required to change by external factors such as regulations and fines, or adapt production practices over time as a result of changing climate conditions. Published version

Chitin and its derivatives are natural biopolymers that are often used as compounds for the control of soilborne plant pathogens. In spite of recent advances in agricultural practices involving chitin amendments, the microbial communities in chitin-amended soils remain poorly known. The objectives of this study were (1) to investigate the bacterial diversity and abundance in an agricultural soil supplemented with chitin that turned disease-suppressive and (2) to assess the emergence of chitinolytic bacteria under conditions of raised soil pH. Amplicon pyrosequencing of soil-extracted DNA based on the 168 rRNA genes was used to characterize the structures of bacterial communities in soil, chitin-amended or not, with native versus raised pH (5.7 vs 8.7), in microcosms and the field. As a result of chitin addition, changes in the relative abundances of Actinobacteria, Proteobacteria and Bacteroidetes were observed in the field soil. A large and significant increase of the relative abundance of Oxalobacteraceae (Betaproteobacteria, Burkholderiales) was found. Within the Oxalobacteraceae, the genera Duganella and Massilia showed large increases. Moreover, responses of the Alpha- and Gammaproteobacteria appeared shortly after the alteration of the soil pH in the microcosms. A significant decrease in the abundance of Actinobacteria was observed in the chitin-amended field soil and in the microcosm at high pH. Overall, the bacterial abundance in soil tended to decrease with the addition of chitin. Two groups, Actinobacteria and Oxalobacteraceae, were found to be most responsive to the amendment. These results enhance the understanding of responses to chitin and possible interactions within bacterial communities in soil that can be correlated to soil disease suppressiveness. (C) 2014 Elsevier Ltd. All rights reserved.

The Cf-5 gene of tomato confers resistance to strains of the fungal pathogen Cladosporium fulvum carrying the avirulence gene Avr5. Although Cf-5 has been cloned, Avr5 has remained elusive. We report the cloning of Avr5 using a combined bioinformatic and transcriptome sequencing approach. RNA-Seq was performed on the sequenced race 0 strain (0WU; carrying Avr5), as well as a race 5 strain (IPO 1979; lacking a functional Avr5 gene) during infection of susceptible tomato. Forty-four in planta–induced C. fulvum candidate effector (CfCE) genes of 0WU were identified that putatively encode a secreted, small cysteine-rich protein. An expressed transcript sequence comparison between strains revealed two polymorphic CfCE genes in IPO 1979. One of these conferred avirulence to IPO 1979 on Cf-5 tomato following complementation with the corresponding 0WU allele, confirming identification of Avr5. Complementation also led to increased fungal biomass during infection of susceptible tomato, signifying a role for Avr5 in virulence. Seven of eight race 5 strains investigated escape Cf-5-mediated resistance through deletion of the Avr5 gene. Avr5 is heavily flanked by repetitive elements, suggesting that repeat instability, in combination with Cf-5-mediated selection pressure, has led to the emergence of race 5 strains deleted for the Avr5 gene.

Assessing the process that gives rise to hybrid pathogens is central to understanding the evolution of emerging plant diseases. Phytophthora ×alni , a pathogen of alder, results from the homoploid hybridization of two related species, Phytophthora uniformis and Phytophthora × multiformis . Describing the genetic characteristics of P . × alni should help us understand how reproductive mechanisms and historical processes shaped the population structure of this emerging hybrid pathogen. The population genetic structure of P . × alni and the relationship with its parental species were investigated using 12 microsatellites and one mitochondrial DNA (mtDNA) marker on a European collection of 379 isolates. Populations of P . × alni were dominated by one multilocus genotype (MLG). The frequency of this dominant MLG increased after the disease emergence together with a decline in diversity, suggesting that it was favored by a genetic mechanism such as drift or selection. Combined microsatellite and mtDNA results confirmed that P . × alni originated from multiple hybridization events that involved different genotypes of the progenitors. Our detailed analyses point to a geographic structure that mirrors that observed for P. uniformis in Europe. The study provides more insights on the contribution of P. uniformis , an invasive species in Europe, to the emergence of Phytophthora -induced alder decline. IMPORTANCE Our study describes an original approach to assess the population genetics of polyploid organisms using microsatellite markers. By studying the parental subgenomes present in the interspecific hybrid P. ×alni , we were able to assess the geographical and temporal structure of European populations of the hybrid, shedding new light on the evolution of an emerging plant pathogen. In turn, the study of the parental subgenomes permitted us to assess some genetic characteristics of the parental species of P. ×alni , P. uniformis , and P . × multiformis , which are seldom sampled in nature. The subgenomes found in P. ×alni represent a picture of the “fossilized” diversity of the parental species.

The choice of soilless growing medium for plant nutrition, growth and support is crucial for improving the eco-sustainability of the production in horticultural systems. As our current understanding of the functional microbial communities inhabiting this ecosystem is still limited, we examined the microbial community development of the two most important growing media (organic and mineral) used in open soilless horticultural systems. We aimed to identify factors that influence community composition over time and to compare the distribution of individual taxa across growing media and their potential functionality. High throughput sequencing analysis revealed a distinctive and stable microbial community in the organic growing medium. Humidity, pH, nitrate-N, ammonium-N and conductivity were uncovered as the main factors associated with the resident bacterial communities. Ammonium-N was correlated with Rhizobiaceae abundance, while potential competitive interactions among both Methylophilaceae and Actinobacteridae with Rhizobiaceae were suggested. Our results revealed that soilless growing media are unique niches for diverse bacterial communities with temporal functional stability, which may possibly impact the resistance to external forces. These differences in communities can be used to develop strategies to move towards a sustainable horticulture with increased productivity and quality.

Plants lack the seemingly unlimited receptor diversity of a somatic adaptive immune system as found in vertebrates and rely on only a relatively small set of innate immune receptors to resist a myriad of pathogens. Here, we show that disease-resistant tomato plants use an efficient mechanism to leverage the limited nonself recognition capacity of their innate immune system. We found that the extracellular plant immune receptor protein Cf-2 of the red currant tomato ( Solanum pimpinellifolium ) has acquired dual resistance specificity by sensing perturbations in a common virulence target of two independently evolved effectors of a fungus and a nematode. The Cf-2 protein, originally identified as a monospecific immune receptor for the leaf mold fungus Cladosporium fulvum , also mediates disease resistance to the root parasitic nematode Globodera rostochiensis pathotype Ro1-Mierenbos. The Cf-2–mediated dual resistance is triggered by effector-induced perturbations of the apoplastic Rcr3 pim protein of S. pimpinellifolium . Binding of the venom allergen-like effector protein Gr-VAP1 of G. rostochiensis to Rcr3 pim perturbs the active site of this papain-like cysteine protease. In the absence of the Cf-2 receptor, Rcr3 pim increases the susceptibility of tomato plants to G. rostochiensis , thus showing its role as a virulence target of these nematodes. Furthermore, both nematode infection and transient expression of Gr-VAP1 in tomato plants harboring Cf-2 and Rcr3 pim trigger a defense-related programmed cell death in plant cells. Our data demonstrate that monitoring host proteins targeted by multiple pathogens broadens the spectrum of disease resistances mediated by single plant immune receptors.

Biopesticides based on living microbes and their bioactive compounds have been researched and promoted as replacements for synthetic pesticides for many years. However, lack of efficacy, inconsistent field performance and high cost have generally relegated them to niche products. Recently, technological advances and major changes in the external environment have positively altered the outlook for biopesticides. Significant increases in market penetration have been made, but biopesticides still only make up a small percentage of pest control products. Progress in the areas of activity spectra, delivery options, persistence of effect and implementation have contributed to the increasing use of biopesticides, but technologies that are truly transformational and result in significant uptake are still lacking.

Interactions between Serratia plymuthica A30 and a blackleg-causing biovar 3 Dickeya sp. were examined. In a potato slice assay, S. plymuthica A30 inhibited tissue maceration caused by Dickeya sp. IPO2222 when co-inoculated at a density at least 10 times greater than that of the pathogen. In glasshouse experiments, population dynamics of the antagonist and of the pathogen in planta were studied by dilution plating and confocal laser scanning microscopy (CLSM) using fluorescent protein-tagged strains. Pathogen-free minitubers were vacuum-infiltrated with DsRed-tagged Dickeya sp. IPO2222 and superficially treated during planting with a water suspension containing GFP-tagged S. plymuthica A30. A30 reduced the blackleg incidence from 55% to 0%. Both the pathogen and the antagonist colonized the seed potato tubers internally within 1 day post-inoculation (dpi). Between 1 and 7 dpi, the population of A30 in tubers increased from 101 to c. 103 CFU g)1 and subsequently remained stable until the end of the experiment (28 dpi). Populations of A30 in stems and roots increased from c. 102 to c. 104 CFU g)1 between 7 and 28 dpi. Dilution plating and CLSM studies showed that A30 decreased the density of Dickeya sp. populations in plants. Dilution plating combined with microscopy allowed the enumeration of strain A30 and its visualization in the vascular tissues of stem and roots and in the pith of roots, as well as its adherence to and colonization of the root surface. The implications of these finding for the use of S. plymuthica A30 as a biocontrol agent are discussed

Early detection of infection is very important for efficient management of Mycosphaerella graminicola leaf blotch. To monitor and quantify the occurrence of this fungus during the growing season, a diagnostic method based on real-time PCR was developed. Standard and real-time PCR assays were developed using SYBR Green chemistry to quantify M. graminicola in vitro or in wheat samples. Microsatellite dinucleotide specific-primers were designed based on microsatellite repeats of sequences present in the genome of M. graminicola. Specificity was checked by analyzing DNA of 55 M. graminicola isolates obtained from different geographical origins. The method appears to be highly specific for detecting M. graminicola, no fluorescent signals were observed from 14 other closely related taxa. Primer (CT) 7 G amplified a specific amplicon of 570 bp from all M. graminicola isolates. The primers did not amplify DNA extracted from 14 other fungal species. The approximate melting temperature (Tm) of the (CT) 7 G primer was 84.2 °C. The detection limit of the real-time PCR assay with the primer sets (CT) 7 G is 10 fg/25 µL, as compared to 10 pg/25 µL using conventional PCR technology. From symptomless leaves, a PCR fragment could be generated two days after inoculation. Both conventional and real-time PCR could successfully detect the fungus from artificially inoculated wheat leaves. However, real-time PCR appeared much more sensitive than conventional PCR. The developed quantitative real-time PCR method proved to be rapid, sensitive, specific, cost-effective and reliable for the identification and quantification of M. graminicola in wheat.

International audience; Here, we describe the development of an oomycete-specific primer pair for amplification of the cytochrome b region in plant pathogenic species that span the order Peronosporales (Phytophthora spp., downy mildews). Because of the high number of variable sites at both inter- and intra-specific levels this marker provides a powerful tool for population genetics and phylogenetic studies in this taxa. We also demonstrate its potential compared with other oomycete-specific mitochondrial markers currently available.

Simultaneous detection and identification of multiple pathogenic microorganisms in complex environmental samples are required in numerous diagnostic fields. Here, we describe the development of a novel, background-free ligation detection (LD) system using a single compound detector probe per target. The detector probes used, referred to as padlock probes (PLPs), are long oligonucleotides containing asymmetric target complementary regions at both their 5′ and 3′ ends which confer extremely specific target detection. Probes also incorporate a desthiobiotin moiety and an internal endonuclease IV cleavage site. DNA samples are PCR amplified, and the resulting products serve as potential targets for PLP ligation. Upon perfect target hybridization, the PLPs are circularized via enzymatic ligation, captured, and cleaved, allowing only the originally ligated PLPs to be visualized on a universal microarray. Unlike previous procedures, the probes themselves are not amplified, thereby allowing a simple PLP cleavage to yield a background-free assay. We designed and tested nine PLPs targeting several oomycetes and fungi. All of the probes specifically detected their corresponding targets and provided perfect discrimination against closely related nontarget organisms, yielding an assay sensitivity of 1 pg genomic DNA and a dynamic detection range of 10 4 . A practical demonstration with samples collected from horticultural water circulation systems was performed to test the robustness of the newly developed multiplex assay. This novel LD system enables highly specific detection and identification of multiple pathogens over a wide range of target concentrations and should be easily adaptable to a variety of applications in environmental microbiology.

A cultivation-based approach was used to determine the in vitro antagonistic potential of soil bacteria towards Rhizoctonia solani AG3 and Fusarium oxysporum f. sp. lini (Foln3). Four composite soil samples were collected from four agricultural sites with previous documentation of disease suppression, located in France (FR), the Netherlands (NL), Sweden (SE) and the United Kingdom (UK). Similarly, two sites from Germany (Berlin, G-BR; and Braunschweig, G-BS) without documentation of disease suppression were sampled. Total bacterial counts were determined by plating serial dilutions from the composite soil samples onto R2A, AGS and King's B media. A total of 1,788 isolates (approximately 100 isolates per medium and site) was screened for antifungal activity, and in vitro antagonists (327 isolates) were found amongst the dominant culturable bacteria isolated from all six soils. The overall proportion of antagonists and the number of isolates with inhibitory activity against F. oxysporum were highest in three of the suppressive soils (FR, NL and SE). Characterization of antagonistic bacteria revealed a high phenotypic and genotypic diversity. Siderophore and protease activity were the most prominent phenotypic traits amongst the antagonists. The composition and diversity of antagonists in each soil was site-specific. Nevertheless, none of the antimicrobial traits of bacteria potentially contributing to soil suppressiveness analyzed in this study could be regarded as specific to a given site. (c) 2007 Elsevier Ltd. All rights reserved.

The abiotic conditions required for eradication of Polymyxa betae, the vector of Beet necrotic yellow vein virus in sugar beet, were investigated. Survival of resting spores of P. betae was determined under aerobic (30 min, 4 days and 21 days) and anaerobic (4 days) conditions under several temperature regimes in a water suspension and in leachate extracted from an aerobic compost heap. In water under aerobic conditions the lethal temperature was 60, 55 and 40°C for exposure times of 30 min, 4 days and 21 days, respectively. The effect of compost leachate and/or anaerobic conditions on survival of P. betae depended on temperature. After incubation for 4 days at 20°C, no significant effects of anaerobic conditions or leachate on the survival of P. betae were found. However, at 40°C for 4 days under anaerobic conditions, survival of P. betae was significantly lower than survival under aerobic conditions in water as well as in leachate. In leachate taken from an aerobic compost heap, aerobically incubated at 40°C for 4 days, survival of P. betae was significantly lower than survival in water at the same temperature. As anaerobic spots are prevalent in aerobic compost heaps, especially during the thermophilic phase, actual inactivation temperatures under composting conditions are likely to be lower than the temperatures we found for eradication in water under aerobic conditions.

The potential of Biological Soil Disinfestation (BSD) to control potato brown rot, caused by Ralstonia solanacearum race 3 biovar 2, was investigated. BSD involves the induction of anaerobic soil conditions by increasing microbial respiration through incorporation of fresh organic amendments (here: grass or potato haulms) and by reducing re-supply of oxygen by covering with airtight plastic sheets. Control treatments were left without cover and amendment, or amended without covering or covered only without amendment. The effect of BSD on survival of R. solanacearum was tested at three different scales: in 1-l glass mesocosms under laboratory conditions, in 1.2-m-diam microplots positioned in an outdoor quarantine field, and in a naturally infested commercial field. Within a few days, anaerobic conditions developed in the BSD-treated soils. In the mesocosm and microplot experiment, anaerobic conditions persisted till the end of the 4-week experimental period. In the field experiment, the period of anaerobiosis was shorter due to birds damaging the plastic cover. In all three experiments, BSD reduced soil populations of R. solanacearum significantly by 92.5% to >99.9% compared to the non-amended and uncovered control treatments. In the field experiment, BSD also resulted in a significant reduction of R. solanacearum survival in potato tubers buried at 15 or 35 cm and in the rapid decomposition of superficially buried potatoes remaining after harvesting, thus destroying an important inoculum reservoir of R. solanacearum. The treatments with grass amendment only or covering with only plastic did not result in anaerobic conditions and did not decrease R. solanacearum populations during the experimental period. PCR-DGGE analyses of 16S-rDNA from soil samples of the various treatments in the mesocosm and microplot experiments revealed that BSD hardly affected bacterial diversity but did result in clear shifts in the composition of the bacterial community. The possible implications of these shifts are discussed. It is concluded that BSD has the potential to strongly decrease soil infestation levels of R. solanacearum and to become an important element in a sustainable and effective management strategy for potato brown rot, especially in areas where the disease is endemic.

Attempting to achieve long-lasting and stable resistance using uniformly deployed rice varieties is not a sustainable approach. The real situation appears to be much more complex and dynamic, one in which pathogens quickly adapt to resistant varieties. To prevent disease epidemics, deployment should be customized and this decision will require interdisciplinary actions. This perspective article aims to highlight the current progress on disease resistance deployment to control bacterial blight in rice. Although the model system rice-Xanthomonas oryzae pv. oryzae has distinctive features that underpin the need for a case-by-case analysis, strategies to integrate those elements into a unique decision tool could be easily extended to other crops. Global Rice Science Partnership (GRiSP) BIll & Melinda Gates Foundation (BMGF) Philippine Rice Information System

Rcr3 and Pip1 are paralogous secreted papain-like proteases of tomato. Both proteases are inhibited by Avr2 from the fungal pathogen Cladosporium fulvum, but only Rcr3 acts as a co-receptor for Avr2 recognition by the tomato Cf-2 immune receptor [ 1, 2, 3 and 4]. Here, we show that Pip1-depleted tomato plants are hyper-susceptible to fungal, bacterial, and oomycete plant pathogens, demonstrating that Pip1 is an important broad-range immune protease. By contrast, in the absence of Cf-2, Rcr3 depletion does not affect fungal and bacterial infection levels but causes increased susceptibility only to the oomycete pathogen Phytophthora infestans. Rcr3 and Pip1 reside on a genetic locus that evolved over 36 million years ago. These proteins differ in surface-exposed residues outside the substrate-binding groove, and Pip1 is 5- to 10-fold more abundant than Rcr3. We propose a model in which Rcr3 and Pip1 diverged functionally upon gene duplication, possibly driven by an arms race with pathogen-derived inhibitors or by coevolution with the Cf-2 immune receptor detecting inhibitors of Rcr3, but not of Pip1.

Agriculture faces the challenge of meeting increasing food demands whilst simultaneously satisfying ever stringent sustainability goals. Taken together with the ever increasing rate of integrated globalisation and other anthropogenic impacts, this challenge is further complicated by climate change. Climate change is indeed increasingly recognised as a considerable risk to agriculture in the European Union, particularly with respect to direct impacts on crop production and yield stability. A major impact threat is the further risk from new and emerging invasive alien species, and potential novel pathogenically aggressive adaptations in existing indigenous pests and pathogens, which, hitherto, have been managed with conventional practices and approaches. The introduction of several exotic pests such as Tuta absoluta, Bemisia tabaci, and Bactrocera fruit flies in Europe points out the changing trend in pathogen adaptation to new regions due to climate change thereby threatening the viability of European crop production. Likewise, slight increases in temperature heighten disease severity caused by indigenous pathogens such as Leptosphaeria maculans, Fusarium graminearum and Dickeya spp. on oilseed rape, cereals and potato, respectively in Europe. Over the last century, there has been an increased global mean temperature by 0.74 degrees C which is projected to rise by 3.4 degrees C by the end of twenty-first century. This raise in temperature has resulted in increased pest pressure in European agriculture through a shift from lower latitudes pole-wards and from lower to higher altitudes. In view of this, the development of anticipatory adaptive strategies, resulting in more resilient cropping systems, is the only alternative to tackle evolving pests under changing climate in order to ensure food security for a global population estimated to reach 9.6 billion by 2050.

The effect of mixed cropping on disease suppressiveness of soils was tested for two cropping systems, Brussels sprouts-barley and triticale-white clover. Disease suppressiveness of field soils was evaluated in bioassays for the soilborne pathogens Rhizoctonia solani, Fusarium oxysporum f. sp. lini, and Gaeumannomyces graminis var. tritici. For both cropping systems, mixed cropping did not enhance disease suppressiveness of the soils. In some cases, soil cropped to barley alone was significantly more suppressive to F. oxysporum f. sp. lini than soils cropped to Brussels sprouts or the mixture of Brussels sprouts and barley. Analyses of the diversity of the indigenous bacterial and fungal microflora by denaturing gradient gel electrophoresis of amplified 16S- and 18S-rDNA fragments, respectively, revealed, in most cases, no significant differences between mixed and mono-cropped soils. In conclusion, in this study, mixed cropping of soils with Brussels sprouts and barley or with triticale and white clover did not enhance microbial diversity or disease suppressiveness of soils to three different soilborne plant pathogens.

Assessment of phytosanitary risks associated with application of composts in agriculture generally has focused on the sanitation (self-heating) phase during composting when most plant pathogens are inactivated due to lethal temperatures. However, a few plant pathogens are heat resistant and they may survive a properly monitored and controlled composting process. To assess the phytosanitary risks associated with compost utilization, several additional factors need to be considered which all relate to a tracing-and-tracking principle. It includes the composition of the original waste and several aspects related to compost utilization. The following parameters are considered to be key-factors: 1) the proportion of host biomass relative to the total quantity of biowaste, 2) the proportion of host infected with a pathogen, 3) the density of infected host material, 4) the proportion of propagules of a pathogen that survived the process, and 5) the threshold density of a pathogen in soil above which disease of the host is expected to develop. While the first two parameters may be rather easy to estimate and information on survival of many plant pathogens can be obtained from the literature, little knowledge exists on the density of the pathogens in host materials or on threshold values. This applies particularly to virus diseases. The phytosanitary risk of several types of plant pathogens is discussed in some detail in this paper. Recommendations are given for testing of a composting process for phytohygienic safety.

Five Botrytis cinerea endopolygalacturonase enzymes (BcPGs) were individually expressed in Pichia pastoris, purified to homogeneity and biochemically characterized. While the pH optima of the five enzymes were similar (approximately pH 4.5) the maximum activity of individual enzymes differed significantly. For hydrolysis of polygalacturonic acid (PGA), the Vmax,app ranged from 10 to 900 U mg1, while the Km,app ranged from 0.16 to 0.6 mg ml1. Although all BcPGs are true endopolygalacturonases, they apparently have different modes of action. PGA hydrolysis by BcPG1, BcPG2 and BcPG4 leads to the transient accumulation of oligomers with DP

The epidemiology of the rust fungus Endophyllum osteospermi was investigated. This rust fungus is considered to be a candidate biological control agent for Chrysanthemoides monilifera ssp. monilifera, which is an invasive alien weed of native vegetation in south-eastern Australia. Between 10 and 20 plants of C. monilifera were marked at each of five sites in the Western Cape Province of South Africa, where both organisms are native. The infection levels and number of witches¿ brooms were determined every 2 months over a 2-year period. Additionally, at three of these sites, the infection levels and number of witches¿ brooms of all bushes in the host population was determined annually over 4 years. The increase in number of witches¿ brooms per bush ranged between 0 and 282 within 1 year, with an average increase per bush of 28 (SE±4.8) in 1993, and 39 (SE±9.2) in 1994. The average rs for all bushes during 1993 was 0.015 month¿1 (SE±0.0041, n=72) and 0.0098 month¿1 (SE±0.0073, n=43) during 1994. When host bushes that either died back or died during the course of each year were excluded, then the average rs during 1993 was 0.023 month¿1 (SE±0.0048, n=45) and 0.0348 month¿1 (SE±0.0106, n=20) during 1994. Under suitable conditions in South Africa, E. osteospermi undergoes epidemic increase within its host plant's populations. This rust fungus should therefore be considered as a suitable candidate biological control agent for use in Australia against C. monilifera ssp. monilifera.

Lysobacter enzymogenes strain 3.1T8 is a potential biocontrol agent of Pythium aphanidermatum (Edson) Fitzp. in cucumber (Cucumis sativa L.). It showed in vitro antagonistic activity, hemolytic activity, and the production of a surface active compound, which decreased in media of increasing strength. One or more low molecular compounds, present in culture filtrates of 3.1T8, caused rapid immobilization of zoospores of P. aphanidermatum and inhibited cyst germination. Bioassays for suppression of root and crown rot were performed with 2-week-old cucumber plants in nutrient solution. Complete disease suppression occurred after addition of a suspension of unwashed cells grown on nutrient-poor R2A, or the filtrate of this suspension, 1 day before inoculation with zoospores. Application of washed cells caused slight suppression in one of two bioassays only. No disease suppression occurred when bacterial inoculum grown on 1/10 TSBA or in 1/10 TSB was used. In two bioassays on cucumber plants grown for 4–5 weeks in 18-L containers, application of R2A-grown cells of 3.1T8 did not cause disease suppression. Density of 3.1T8, monitored using immunofluorescence colony staining, decreased at least 100-fold during these experiments to approximately log 3–4 cfu ml-1 of nutrient solution and log 5–6 cfu g-1 roots. Overall, the findings indicated a strong influence of nutritional resources on production of antifungal compounds by strain 3.1T8. The lack of disease suppression in hydroculture containers suggested that in situ production of the compounds was insufficient or absent, although the bacteria persisted in the nutrient solution and on the roots of cucumber. [KEYWORDS: Lysobacter enzymogenes ; Pythium aphanidermatum ; Cucumis sativa L. ; Hydroponics ; Rockwool ; Biosurfactant ; Antibiotic ; Root colonization ; Quorum sensing]

Conventional agriculture has had major environmental impacts, in particular with respect to soil degradation. Soil structure, fertility, microbial and faunal biodiversity have declined, and root diseases are common unless genetic resistance, soil fumigation and/or seed treatments are used. Primarily for environmental reasons and increasing demands for safe and healthy food from the public, farmers have switched over to organic production at an increasing rate. During a transition period of about 5 years, organic farmers may face problems with yield losses and pest or disease problems. However, in well-managed, long-term organic farms, soilborne diseases need not be a problem. Several studies in which disease severity was compared in organic and conventional farming systems (or with soils from those systems) showed that root diseases are generally less severe in organically than conventionally managed soils. The reasons for reduced root disease severity have seldom been investigated, although relationships with nitrogen supply or microbial diversity have sometimes been found. Crop protection in organic farming is generally not directed at controlling particular pathogens or pests but at management of the environment so that plants are able to withstand potential attacks. Resistant cultivars adapted to the local conditions are in demand among organic farmers. However, the main practices that contribute to disease control are long, balanced rotations, organic amendments and reduced tillage, all geared towards maintenance of the soil organic matter content and fertility. Organic farmers can make use of biological control agents and natural toxic compounds in plant extracts. However, these practices are methods of last resort.

Vascular wilts caused by Verticillium spp. are destructive plant diseases affecting hundreds of hosts. Only a few Verticillium spp. are causal agents of vascular wilt diseases, of which V. dahliae is the most notorious pathogen, and several V. dahliae genomes are available. In contrast, V. tricorpus is mainly known as a saprophyte and causal agent of opportunistic infections. Based on a hybrid approach that combines second and third generation sequencing, a near-gapless V. tricorpus genome assembly was obtained. With comparative genomics, we sought to identify genomic features in V. dahliae that confer the ability to cause vascular wilt disease. Unexpectedly, both species encode similar effector repertoires and share a genomic structure with genes encoding secreted proteins clustered in genomic islands. Intriguingly, V. tricorpus contains significantly fewer repetitive elements and an extended spectrum of secreted carbohydrate- active enzymes when compared with V. dahliae. In conclusion, we highlight the technical advances of a hybrid sequencing and assembly approach and show that the saprophyte V. tricorpus shares many hallmark features with the pathogen V. dahliae.

The poplar rust fungus Melampsora larici-populina causes significant yield reduction and severe economic losses in commercial poplar plantations. After several decades of breeding for qualitative resistance and subsequent breakdown of the released resistance genes, breeders now focus on quantitative resistance, perceived to be more durable. But quantitative resistance also can be challenged by an increase of aggressiveness in the pathogen. Thus, it is of primary importance to better understand the genetic architecture of aggressiveness traits. To this aim, our goal is to build a genetic linkage map for M. larici-populina in order to map quantitative trait loci related to aggressiveness. First, a large progeny of M. larici-populina was generated through selfing of the reference strain 98AG31 (which genome sequence is available) on larch plants, the alternate host of the poplar rust fungus. The progeny's meiotic origin was validated through a segregation analysis of 115 offspring with 14 polymorphic microsatellite markers, of which 12 segregated in the expected 1:2:1 Mendelian ratio. A microsatellite-based linkage disequilibrium analysis allowed us to identify one potential linkage group comprising two scaffolds. The whole genome of a subset of 47 offspring was resequenced using the Illumina HiSeq 2000 technology at a mean sequencing depth of 6X. The reads were mapped onto the reference genome of the parental strain and 144,566 SNPs were identified across the genome. Analysis of distribution and polymorphism of the SNPs along the genome led to the identification of 2580 recombination blocks. A second linkage disequilibrium analysis, using the recombination blocks as markers, allowed us to group 81 scaffolds into 23 potential linkage groups. These preliminary results showed that a high-density linkage map could be constructed by using high-quality SNPs based on low-coverage resequencing of a larger number of M. larici-populina offspring.

Microorganisms play essential roles in almost every environment on earth. For instance, microbes decompose organic material, or establish symbiotic relationships that range from pathogenic to mutualistic. Symbiotic relationships have been particularly well studied for microbial plant pathogens and have emphasized the role of effectors; secreted molecules that support host colonization. Most effectors characterized thus far play roles in deregulation of host immunity. Arguably, however, pathogens not only deal with immune responses during host colonization, but also encounter other microbes including competitors, (myco)parasites and even potential co-operators. Thus, part of the effector catalog may target microbiome co-inhabitants rather than host physiology.

Melampsora larici-populina is a fungal pathogen responsible for foliar rust disease on poplar trees, which causes damage to forest plantations worldwide, particularly in Northern Europe. The reference genome of the isolate 98AG31 was previously sequenced using a whole genome shotgun strategy, revealing a large genome of 101 megabases containing 16,399 predicted genes, which included secreted protein genes representing poplar rust candidate effectors. In the present study, the genomes of 15 isolates collected over the past 20 years throughout the French territory, representing distinct virulence profiles, were characterized by massively parallel sequencing to assess genetic variation in the poplar rust fungus. Comparison to the reference genome revealed striking structural variations. Analysis of coverage and sequencing depth identified large missing regions between isolates related to the mating type loci. More than 611,824 single-nucleotide polymorphism (SNP) positions were uncovered overall, indicating a remarkable level of polymorphism. Based on the accumulation of non-synonymous substitutions in coding sequences and the relative frequencies of synonymous and non-synonymous polymorphisms (i.e., PN/PS ), we identify candidate genes that may be involved in fungal pathogenesis. Correlation between non-synonymous SNPs in genes encoding secreted proteins (SPs) and pathotypes of the studied isolates revealed candidate genes potentially related to virulences 1, 6, and 8 of the poplar rust fungus.

Crop pathogens are notorious for their rapid adaptation to their host. We still know little about the evolution of their life cycles and whether there might be trade-offs between fitness components, limiting the evolutionary potential of these pathogens. In this study, we explored a trade-off between spore production capacity and latent period in Puccinia triticina, a fungal pathogen causing leaf rust on wheat. Using a simple multivariate (manova) technique, we showed that the covariance between the two traits is under shared control of host and pathogen, with contributions from host genotype (57%), pathogen genotype (18.4%) and genotype × genotype interactions (12.5%). We also found variation in sign and strength of genetic correlations for the pathogen, when measured on different host varieties. Our results suggest that these important pathogen life-history traits do not freely respond to directional selection and that precise evolutionary trajectories are contingent on the genetic identity of the interacting host and pathogen.

Fungal plant pathogens secrete effector molecules to establish disease on their hosts, and plants in turn use immune receptors to try to intercept these effectors. The tomato immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and Verticillium albo-atrum , but the corresponding Verticillium effector remained unknown thus far. By high-throughput population genome sequencing, a single 50-Kb sequence stretch was identified that only occurs in race 1 strains, and subsequent transcriptome sequencing of Verticillium -infected Nicotiana benthamiana plants revealed only a single highly expressed ORF in this region, designated Ave1 (for A virulence on Ve1 tomato ). Functional analyses confirmed that Ave1 activates Ve1-mediated resistance and demonstrated that Ave1 markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis . Interestingly, Ave1 is homologous to a widespread family of plant natriuretic peptides. Besides plants, homologous proteins were only found in the bacterial plant pathogen Xanthomonas axonopodis and the plant pathogenic fungi Colletotrichum higginsianum , Cercospora beticola , and Fusarium oxysporum f. sp. lycopersici . The distribution of Ave1 homologs, coincident with the presence of Ave1 within a flexible genomic region, strongly suggests that Verticillium acquired Ave1 from plants through horizontal gene transfer. Remarkably, by transient expression we show that also the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1 -mediated resistance. In line with this observation, Ve1 was found to mediate resistance toward F. oxysporum in tomato, showing that this immune receptor is involved in resistance against multiple fungal pathogens.