Short-term effects of soil solarization in suppressing Calonectria microsclerotia

Background and aims

Calonectria species have been reported as devastating pathogens mostly on horticultural and forest crops worldwide. Since these pathogens represent a serious threat for the nursery production, the aim of this study was to investigate on the short-term potential of soil solarization for eradicating Calonectria microsclerotia.

Methods

Twenty Calonectria isolates collected in Italy from different hosts and locations were identified by using DNA sequencing of β-tubulin. The effect of thermal regimes and innovative solarizing films on the soil survival of Calonectria microsclerotia was evaluated through time at different sampling periods in growth chamber and greenhouse experiments.

Results

Eleven and nine isolates were identified as Calonectria pauciramosa and Calonectria polizzii, respectively. No viable Calonectria inoculum was recovered after 12 days from all solarized plots inside ethylene-tetrafluoroethylene (ETFE) greenhouse and at 15-cm depth from solarized plots inside ethylene-vinyl-acetate (EVA) greenhouse. Under EVA cover, solarization killed C. pauciramosa microsclerotia within 9 and 17 d at 15- and 30-cm depths in soil, respectively, whereas no viable inoculum was retrieved within 6 and 12 days from solarized plots inside ETFE greenhouse.

Conclusions

This paper demonstrates that short-term soil solarization is effective for Calonectria microsclerotia suppression in nurseries, and shows that ETFE film as well as other innovative materials could improve this technique.     

Florigenic Acid from fungal culture

The extraction procedures which have been successfully employed in the preparation of a florigenic principle from the tissues of Xanthium, are applicable to the derivation of an entity of similar activity from Calonectria culture. The Xanthium principle is acedic, with pK_a values characteristic of a carboxylic acid (6). Although definitive chemical comparisons have not been completed, the extraction and solvent partition procedures that have been applicable to the extraction of the active entity from higher plant tissues have yielded florigenic preparations from fungal culture. The chemical principle from higher plant tissue may be the same or similar to the florigenic agent of Calonectria.

The many responses of higher plants to growth regulators produced by micro-organisms are well known. The presence of a flower-producing principle from Calonectria (Fusarium) rigidiuscula parallels closely the pattern exhibited by those fungal species capable of the production of auxin and gibberellins.

     

Calonectria leaf blight of Eucalyptus: A global review

Calonectria (= Cylindrocladium), an internationally recognised fungal ascomycete genus, causes significant diseases on numerous crops of forestry, horticultural and agricultural interest. Eucalyptus, an economically important multipurpose plantation forestry species, is severely threatened by Calonectria leaf blight (CLB) disease in Brazil, Australia, China, Indonesia, Vietnam and India. The disease causes serious mortality in the nurseries/plantations and yield losses due to defoliation. Since the first report of Calonectria in 1867, efforts have been made for the identification and characterisation of this vastly diverse hypocrealean genus. Currently, the genus Calonectria harbours 131 species grouped within 11 species complexes, and broadly categorised into the Prolate and Sphaero-Naviculate groups. Globally, 35 Calonectria species distributed in seven species complexes cause CLB of Eucalyptus, out of which 29 belong to the Prolate group and five to the Sphaero-Naviculate group. Likewise, 28 Calonectria species distributed in eight species complexes have been reported in association with infected Eucalyptus tissues and plantation soil, however, their pathogenicity remains to be determined. Given the importance of CLB of Eucalyptus, this review compiles its global impact, shifts in taxonomy and nomenclature of Calonectria, pathogenesis mechanism, defence response of Eucalyptus upon infection and disease management. This study identifies and provides direction to crucial areas of future research on CLB which is severely impacting Eucalyptus plantations globally.     

Phylogenetic relationships and spatial distributions of putative fungal pathogens of seedlings across a rainfall gradient in Panama

Despite their importance in structuring plant communities, the identities and spatial distributions of the pathogens impacting wild plant communities are largely unknown. To advance our knowledge of plant-pathogen interactions in tropical forests, I identified likely fungal pathogens from forest sites across a rainfall gradient in Panama and compared the communities of fungi inhabiting a wetter, Atlantic and a drier, Pacific forest (∼45 km apart). Seedlings with symptoms of pathogen attack were collected and fungi were isolated from the symptomatic tissue. Based on internal transcribed spacer region sequences, I assigned the fungal isolates to operational taxonomic units (OTUs) and estimated their taxonomic placements. I observed 28 OTUs (defined by 95% sequence similarity); primarily, the genera Mycoleptodiscus, Glomerella, Bionectria, Diaporthe, and Calonectria. The wetter, Atlantic and drier, Pacific forest sites shared 29% of observed and 56% of non-singleton fungal OTUs, suggesting that, in these forests, the common fungal pathogens of seedlings are relatively widespread, habitat generalists.     

The use of mutant and engineered microbial agents for biological control of plant diseases caused by Pythium: Achievements versus challenges

Pythium species are devasting pathogens causing major crop losses, e.g., damping-off in sugar beet caused by Pythium ultimum and root-rot of tomato caused by Pythium aphanidermatum. The use of natural antagonistic microorganisms is a promising environment-friendly approach to control Pythium-caused plant diseases. There are several examples of biocontrol of diseases caused by Pythium species but the application of bioeffectors (biological control agents) is limited for various reasons, including the restricted amount of gene-modification based biotechnological progress. The regulations in many countries prevent genetically modified bioeffectors from being routinely deployed in field conditions. Our two connected aims in this review are (1) to compile and assess achievements in genetic modification of bioeffectors which have been tested for parasitism or antagonism towards a Pythium plant pathogen or biocontrol of a plant disease caused by a Pythium species, and (2) discuss how a better performing bioeffector could be engineered to improve biocontrol of Pythium-caused plant diseases. We focus on the role of seven key mechanisms: cellulases, carbon catabolite de-repression, glycosylation, reactive oxygen species, chitin re-modelling, proteases, and toxic secondary metabolites. Genetic modifications of bioeffectors include gene deletion and overexpression, as well as the replacement of promoter elements to tune the gene expression to the presence of the pathogen. Gene-modifications are limited to fungal and bacterial bioeffectors due to the difficulty of gene modification in oomycete bioeffectors such as Pythium oligandrum. We assess how previous gene modifications could be combined and what other gene modification techniques could be introduced to make improved bioeffectors for Pythium-caused plant diseases. The broad host-range of Pythium spp. suggests engineering improved antagonistic traits of a bioeffector could be more effective than engineering plant-mediated traits i.e., engineer a bioeffector to antagonise a plant pathogen in common with multiple plant hosts rather than prime each unique plant host.     

Molecular Inversion Probe: A New Tool for Highly Specific Detection of Plant Pathogens

Highly specific detection methods, capable of reliably identifying plant pathogens are crucial in plant disease management strategies to reduce losses in agriculture by preventing the spread of diseases. We describe a novel molecular inversion probe (MIP) assay that can be potentially developed into a robust multiplex platform to detect and identify plant pathogens. A MIP has been designed for the plant pathogenic fungus Fusarium oxysporum f.sp. conglutinans and the proof of concept for the efficiency of this technology is provided. We demonstrate that this methodology can detect as little as 2.5 ng of pathogen DNA and is highly specific, being able to accurately differentiate Fusarium oxysporum f.sp. conglutinans from other fungal pathogens such as Botrytis cinerea and even pathogens of the same species such as Fusarium oxysporum f.sp. lycopersici. The MIP assay was able to detect the presence of the pathogen in infected Arabidopsis thaliana plants as soon as the tissues contained minimal amounts of pathogen. MIP methods are intrinsically highly multiplexable and future development of specific MIPs could lead to the establishment of a diagnostic method that could potentially screen infected plants for hundreds of pathogens in a single assay.     

Use of endophytes as biocontrol agents

Plant diseases, caused by various microorganisms, including viruses, bacteria, fungi, protozoa and nematodes, affect agricultural practices and result in significant crop losses. Fungal pathogens are the major cause of plant diseases and infect most plants. Agrochemicals play a significant role in plant disease management to ensure a sustainable and productive agricultural system. However, the intensive use of chemicals has adverse effects on humans and ecosystem functioning and also reduces agricultural sustainability. A sustainable agriculture is achieved through reduction or elimination of fertilizers and agrochemicals, resulting in minimal impact to the environment. Recently, the use of antagonistic endophytes as biocontrol agents is drawing special attention as an attractive option for management of some plant diseases, resulting in minimal impact to the environment. Endophytes that resides asymptomatically within a plant, have the potential to provide a source of candidate strains for potential biocontrol applications. This review addresses biocontrol methods using endophytic fungi such as Colletotrichum, Cladosporium, Fusarium, Pestalotiopsis and Trichoderma species as an attractive option for management of some plant diseases. Potential endophytes are screened in vitro and in vivo to test their antagonistic actions by different mechanisms, including mycoparasitism, production of lytic enzymes and/or antibiotics and induction of plant defenses. Currently, efforts are being made to commercialize these biocontrol agents. A continued research pipeline consisting of screening, in vitro and in vivo testing, biomass production and commercialization of endophytes as biocontrol agents may contribute to sustainable agriculture.     

Potential of Lecanicillium spp. for management of insects, nematodes and plant diseases

Fungi in the genus Lecanicillium (formerly classified as the single species Verticillium lecanii) are important pathogens of insects and some have been developed as commercial biopesticides. Some isolates are also active against phytoparasitic nematodes or fungi. Lecanicillium spp. use both mechanical forces and hydrolytic enzymes to directly penetrate the insect integument and the cell wall of the fungal plant pathogen. In addition to mycoparasitism of the plant pathogen, the mode of action is linked to colonization of host plant tissues, triggering an induced systemic resistance. Recently it was demonstrated that development of Lecanicillium hybrids through protoplast fusion may result in strains that inherit parental attributes, thereby allowing development of hybrid strains with broader host range and other increased benefits, such as increased viability. Such hybrids have demonstrated increased virulence against aphids, whiteflies and the soybean cyst nematode. Three naturally occurring species of Lecanicillium, L. attenuatum, L. longisporum, and an isolate that could not be linked to any presently described species based on rDNA sequences have been shown to have potential to control aphids as well as suppress the growth and spore production of Sphaerotheca fuliginea, the causal agent of cucumber powdery mildew. These results suggest that strains of Lecanicillium spp. may have potential for development as a single microbial control agent effective against several plant diseases, pest insects and plant parasitic nematodes due to its antagonistic, parasitic and disease resistance inducing characteristics. However, to our knowledge, no Lecanicillium spp. have been developed for control of phytopathogens or phytoparasitic nematodes.     

内生真菌感染对宿主羊草抗病性的影响

以感染内生真菌的天然禾草羊草为实验材料,通过体外纯培养条件下的内生真菌、感染内生真菌的离体叶片和在体叶片对3种病原菌的抑菌实验,以探讨内生真菌对宿主植物羊草在抗病性方面的贡献。结果表明:体外纯培养条件下,分离自羊草的内生真菌Epichlobromicola对新月弯孢(Curvularia lunata)、根腐离蠕孢(Bipolaris sorokiniana)和枝孢霉(Cladosporium sp.)这3种病原菌都具有抑制作用,抑菌率分别达56.22%,46.93%和45.15%,且内生真菌培养滤液可以有效抑制这3种病原菌的孢子萌发,平均萌发率分别为30.4%,15.7%和16.4%;宿主植物叶片在离体条件下,内生真菌感染可以有效降低羊草叶片受C.lunata和C.sp.侵染后的病斑数或病斑长度,但对B.sorokiniana不起作用,甚至提高了叶片的病斑数及病斑长度,而离体叶片提取液对不同病原菌均有不同程度的抑制作用;在体条件下,内生真菌均可以通过降低叶片病斑数来增强羊草植株对这3种病原菌的抗性。由此看来,内生真菌E.bromicola对宿主植物羊草在抗病原菌侵染方面有一定的增益作用。     

Developing a taxonomic identification system of Phytophthora species based on microsatellites

BackgroundPhytophthora is the most important genus of the Oomycete plant pathogens. Nowadays, there are 117 described species in this genus, most of them being primary invaders of plant tissues. The different species are causal agents of diseases in a wide range of crops and plants in natural environments. In order to develop control strategies against Phytophthoraspecies, it is important to know the biology, ecology and evolutionary processes of these important pathogens.AimsThe aim of this study was to propose and validate a low cost identification system for Phytophthora species based on a set of polymorphic microsatellite (SSRs) markers.MethodsThirty-three isolates representing Phytophthora infestans, Phytophthora andina, Phytophthora sojae, Phytophthora cryptogea, Phytophthora nicotianae, Phytophthora capsici and Phytophthora cinnamomi species were obtained, and 13 SSRs were selected as potentially transferable markers between these species. Amplification conditions, including annealing temperatures, were standardized for several markers.ResultsA subset of these markers amplified in all species, showing species-specific alleles.ConclusionsThe adaptability and impact of the identification system in Colombia, an Andean agricultural country where different Phytophthora species co-exist in the same or in several hosts grown together, are discussed.     

Antiviral potential of Bulgarian medicinal plants

Medicinal plants have been widely used to treat a variety of infectious and non-infectious diseases. Bulgarian flora includes 4,300 plant species, over 500 of which are rare or endemic to the country or the Balkan region. The aim of the present work is to summarize comprehensively the investigations on the antiviral activity of Bulgarian medicinal plants from the past three decades. The effect of different extracts derived from in vitro propagated plants has been examined as well. The phytochemical composition and its influence on specific steps of the viral life cycle have been discussed in this paper. The review includes the following families: Amaryllidaceae, Fabaceae, Geraniaceae, Lamiaceae, Onagraceae, Ranunculaceae, Rosaceae, Scrophulariaceae and Rhodophyta. Special attention has been paid to viruses as important human pathogens.     

An ecological framework for understanding the roles of Epichloë endophytes on plant defenses against fungal diseases

Plants harbor a wide diversity of microorganisms in their tissues. Some of them have a long co-evolutionary history with their hosts, likely playing a pivotal role in regulating the plant interaction with other microbes such as pathogens. Some cool-season grasses are symbiotic with Epichloë fungal endophytes that grow symptomless and systemically in aboveground tissues. Among the many benefits that have been ascribed to endophytes, their role in mediating plant interactions with pathogens has been scarcely developed. Here, we explored the effects of Epichloë fungal endophytes on the interaction of host grasses with fungal pathogens. We made a meta-analysis that covered a total of 18 host grass species, 11 fungal endophyte species, and 22 fungal pathogen species. We observed endophyte-mediated negative effects on pathogens in vitro and in planta. Endophyte negative effects on pathogens were apparent not only in laboratory but also in greenhouse and field experiments. Epichloë fungal endophytes had negative effects on pathogen growth and spores' germination. On living plants, endophytes reduced both severity and incidence of the disease as well as colonization and subsequent infection of seeds. Symbiosis with endophytes showed an inhibitory effect on debilitator and killer pathogens, but not on castrators, and this effect did not differ among biotrophic or necrotrophic lifestyles. We found that this protection can be direct through the production of fungistatic compounds, the competition for a common resource, or the induction of plant defenses, and indirect associated with endophyte-generated changes in the abiotic or the biotic environment. Several mechanisms operate simultaneously and contribute differentially to the reduction of disease within grass populations.     

The complex interactions between host immunity and non-biotrophic fungal pathogens of wheat leaves

Significant progress has been made in elucidating the mechanisms used by plants to recognize pathogens and activate “immune” responses. A “first line” of defense can be triggered through recognition of conserved Pathogen or Microbe Associated Molecular Patterns (PAMPs or MAMPs), resulting in activation of basal (or non-host) plant defenses, referred to as PAMP-triggered immunity (PTI). Disease resistance responses can also subsequently be triggered via gene-for-gene type interactions between pathogen avirulence effector genes and plant disease resistance genes (Avr-R), giving rise to effector triggered immunity (ETI). The majority of the conceptual advances in understanding these systems have been made using model systems, such as Arabidopsis, tobacco, or tomato in combination with biotrophic pathogens that colonize living plant tissues. In contrast, how these disease resistance mechanisms interact with non-biotrophic (hemibiotrophic or necrotrophic) fungal pathogens that thrive on dying host tissue during successful infection, is less clear. Several lines of recent evidence have begun to suggest that these organisms may actually exploit components of plant immunity in order to infect, successfully colonize and reproduce within host tissues. One underlying mechanism for this strategy has been proposed, which has been referred to as effector triggered susceptibility (ETS). This review aims to highlight the complexity of interactions between plant recognition and defense activation towards non-biotrophic pathogens, with particular emphasis on three important fungal diseases of wheat (Triticum aestivum) leaves.     

In Vitro Activity of Glucosinolates and Their Degradation Products against Brassica-Pathogenic Bacteria and Fungi

Glucosinolates (GSLs) are secondary metabolites found in Brassica vegetables that confer on them resistance against pests and diseases. Both GSLs and glucosinolate hydrolysis products (GHPs) have shown positive effects in reducing soil pathogens. Information about their in vitro biocide effects is scarce, but previous studies have shown sinigrin GSLs and their associated allyl isothiocyanate (AITC) to be soil biocides. The objective of this work was to evaluate the biocide effects of 17 GSLs and GHPs and of leaf methanolic extracts of different GSL-enriched Brassica crops on suppressing in vitro growth of two bacterial (Xanthomonas campestris pv. campestris and Pseudomonas syringae pv. maculicola) and two fungal (Alternaria brassicae and Sclerotinia scletoriorum) Brassica pathogens. GSLs, GHPs, and methanolic leaf extracts inhibited the development of the pathogens tested compared to the control, and the effect was dose dependent. Furthermore, the biocide effects of the different compounds studied were dependent on the species and race of the pathogen. These results indicate that GSLs and their GHPs, as well as extracts of different Brassica species, have potential to inhibit pathogen growth and offer new opportunities to study the use of Brassica crops in biofumigation for the control of multiple diseases.     

Molecular Profiling of the Phytophthora plurivora Secretome: A Step towards Understanding the Cross-Talk between Plant Pathogenic Oomycetes and Their Hosts

The understanding of molecular mechanisms underlying host–pathogen interactions in plant diseases is of crucial importance to gain insights on different virulence strategies of pathogens and unravel their role in plant immunity. Among plant pathogens, Phytophthora species are eliciting a growing interest for their considerable economical and environmental impact. Plant infection by Phytophthora phytopathogens is a complex process coordinated by a plethora of extracellular signals secreted by both host plants and pathogens. The characterization of the repertoire of effectors secreted by oomycetes has become an active area of research for deciphering molecular mechanisms responsible for host plants colonization and infection. Putative secreted proteins by Phytophthora species have been catalogued by applying high-throughput genome-based strategies and bioinformatic approaches. However, a comprehensive analysis of the effective secretome profile of Phytophthora is still lacking. Here, we report the first large-scale profiling of P. plurivora secretome using a shotgun LC-MS/MS strategy. To gain insight on the molecular signals underlying the cross-talk between plant pathogenic oomycetes and their host plants, we also investigate the quantitative changes of secreted protein following interaction of P. plurivora with the root exudate of Fagus sylvatica which is highly susceptible to the root pathogen. We show that besides known effectors, the expression and/or secretion levels of cell-wall-degrading enzymes were altered following the interaction with the host plant root exudate. In addition, a characterization of the F. sylvatica root exudate was performed by NMR and amino acid analysis, allowing the identification of the main released low-molecular weight components, including organic acids and free amino acids. This study provides important insights for deciphering the extracellular network involved in the highly susceptible P. plurivora-F. sylvatica interaction.     

Plant-Associated Symbiotic Burkholderia Species Lack Hallmark Strategies Required in Mammalian Pathogenesis

Burkholderia is a diverse and dynamic genus, containing pathogenic species as well as species that form complex interactions with plants. Pathogenic strains, such as B. pseudomallei and B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic pathogens, infecting humans or animals with a compromised immune system. Although some of the opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the risk of infection to infants, the elderly, and people who are immunocompromised has not only resulted in a restriction on their use, but has also limited the application of non-pathogenic, symbiotic species, several of which nodulate legume roots or have positive effects on plant growth. However, recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct lineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts. A number of environmental strains that promote plant growth or degrade xenobiotics are also included in the symbiotic lineage. Many of these species have the potential to enhance agriculture in areas where fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted by climate change. Here we address the pathogenic potential of several of the symbiotic Burkholderia strains using bioinformatics and functional tests. A series of infection experiments using Caenorhabditis elegans and HeLa cells, as well as genomic characterization of pathogenic loci, show that the risk of opportunistic infection by symbiotic strains such as B. tuberum is extremely low.     

Mycoparasitism studies of Trichoderma species against three phytopathogenic fungi: evaluation of antagonism and hydrolytic enzyme production

Trichoderma spp. are used for biocontrol of several plant pathogens. However, their efficient interaction with the host needs to be accompanied by production of secondary metabolites and cell wall-degrading enzymes. Three parameters were evaluated after interaction between four Trichoderma species and plant-pathogenic fungi: Fusarium solani, Rhizoctonia solani and Sclerotinia sclerotiorum. Trichoderma harzianum and T. asperellum were the most effective antagonists against the pathogens. Most of the Trichoderma species produced toxic volatile metabolites, having significant effects on growth and development of the plant pathogens. When these species were grown in liquid cultures with cell walls from these plant pathogens, they produced and secreted β-1,3-glucanase, NAGAse, chitinase, acid phosphatase, acid proteases and alginate lyase.     

Identification and Characterization of Fungal Pathogens Associated with Boxwood Diseases in the Republic of Korea

Boxwood is a representative ornamental shrub that is widely used in landscaping horticulture. After pruning, damaged leaves or stems of boxwoods are unavoidably vulnerable to infection by various plant pathogens. Several boxwood diseases caused by fungi, such as Volutella blight and Macrophoma leaf spot, have been reported worldwide including Republic of Korea. In this study, we isolated and identified fungal pathogens of boxwood diseases that occurred in Korea and characterized their morphological and taxonomic characteristics. Boxwood samples showing blight symptoms were collected in Seoul, Republic of Korea, and the putative fungal pathogens Pseudonectria buxi, P. foliicola, and Neofusicoccum buxi were successfully identified. Investigation of the morphological features of the field isolates, including mycelial growth and conidial morphology, and phylogenetic analysis of multiple DNA barcode loci revealed that there were some morphological and genetic variations among isolates, but all of the analyzed isolates were closely related to the corresponding reference strains. We also found that P. foliicola strains were more virulent than P. buxi, and the N. buxi strains isolated in this study were weak pathogens or saprophytes. The results of our study will contribute to the development of control strategies for boxwood diseases caused by fungi and accelerate research on the complex ecology of boxwood diseases.