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Structural biology has the potential to illuminate the evolution of pathogen effectors and their commonalities that cannot be readily detected at the primary sequence level. Recent breakthroughs in protein structure modeling have demonstrated the feasibility to predict the protein folds without depending on homologous templates. These advances enabled a genome-wide computational structural biology approach to help understand proteins based on their predicted folds. In this study, we employed structure prediction methods on the secretome of the destructive fungal pathogen Magnaporthe oryzae. Out of 1,854 secreted proteins, we predicted the folds of 1,295 proteins (70%). We showed that template-free modeling by TrRosetta captured 514 folds missed by homology modeling, including many known effectors and virulence factors, and that TrRosetta generally produced higher quality models for secreted proteins. Along with sensitive homology search, we employed structure-based clustering, defining not only homologous groups with divergent members but also sequence-unrelated structurally analogous groups. We demonstrate that this approach can reveal new putative members of structurally similar MAX effectors and novel analogous effector families present in M. oryzae and possibly in other phytopathogens. We also investigated the evolution of expanded putative ADP-ribose transferases with predicted structures. We suggest that the loss of catalytic activities of the enzymes might have led them to new evolutionary trajectories to be specialized as protein binders. Collectively, we propose that computational structural genomics approaches can be an integral part of studying effector biology and provide valuable resources that were inaccessible before the advent of machine learning-based structure prediction. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Endophytes associated with plants have the property to produce active biomolecules with their possible applications in agro-industrial sectors. This study provides a project work on analyzing various activities of fungal endophytes isolated from Swertia chirayita of Sikkim Himalayan region. Among several fungal endophytes screened, isolate UTCRF6 was found most active with the secretion of enzymes protease, cellulase, amylase and chitinase, as well as other metabolites Indoleacetic acid and siderophores. This endophyte was found active in restricting the growth of phyto-pathogens, including strains of Fusarium solani, Colletotrichum gloeosporioides, Alternaria alternata, Pestalotiopsis theae and Sclerotinia sclerotiorum. Morphological and molecular studies of this endophytic fungus showed similarity with Penicillium citrinum. Graphic abstract

Plant‐pathogenic fungi are a significant threat to economic and food security worldwide. Novel protection strategies are required and therefore it is critical we understand the mechanisms by which these pathogens cause disease. Virulence factors and pathogenicity genes have been identified, but in many cases their roles remain elusive. It is becoming increasingly clear that gene regulation is vital to enable plant infection and transcription factors play an essential role. Efforts to determine their regulatory functions in plant‐pathogenic fungi have expanded since the annotation of fungal genomes revealed the ubiquity of transcription factors from a broad range of families. This review establishes the significance of transcription factors as regulatory elements in plant‐pathogenic fungi and provides a systematic overview of those that have been functionally characterized. Detailed analysis is provided on regulators from well‐characterized families controlling various aspects of fungal metabolism, development, stress tolerance, and the production of virulence factors such as effectors and secondary metabolites. This covers conserved transcription factors with either specialized or nonspecialized roles, as well as recently identified regulators targeting key virulence pathways. Fundamental knowledge of transcription factor regulation in plant‐pathogenic fungi provides avenues to identify novel virulence factors and improve our understanding of the regulatory networks linked to pathogen evolution, while transcription factors can themselves be specifically targeted for disease control. Areas requiring further insight regarding the molecular mechanisms and/or specific classes of transcription factors are identified, and direction for future investigation is presented., Transcription factors in plant‐pathogenic fungi are systematically reviewed. This includes their classification and a current state of the functional research into their diverse regulatory roles pertaining to fungal virulence.

Blackberry (Rubus spp.) is a shrub plant of the Rosaceae, which fruits are used for fresh markets or processing. Blackberry cultivation has increased in Brazil and has turned into an alternative for family farmers. Among the significant diseases that can occur in plantations, there is rust, caused by phytopathogenic fungus species: Gymnoconia nitens (orange rust), Kuehneola uredinis (cane and leaf rust) and Phragmidium violaceum (blackberry rust). This research project was conducted to identify the fungi specie causative of rust occurred on experimental blackberry fields, in Diamantina, state of Minas Gerais, Brazil. Infected blackberry leaves with symptoms of rust fungi and full spores yellow were collected. Infection was limited to undersurfaces of the leaves and reddening occurred on corresponding upper areas. No stem or fruit infection occurred on the infected plants. Morphological analyzes of the microstructures (sorus and spores) were carried out under optical microscopy and scanning electron microscopy (SEM). It was observed uredinia with urediniospores uniformly echinulate golden-yellow and telia obtain with teliospores smooth hyaline, mostly four celled and with a short rounded apical papilla. The rust was identified as Kuehneola uredinis (Link) Arthur. This is the first record for Brazil. Morphological descriptions, illustrations of the microstructures, examined material, geographic distribution and taxonomic comments are provided for this species.

Key message Modified pEAQ-HT-DEST1 vectors were used for agroinfiltration in legumes. We demonstrate protein expression and export in pea, lentil, and faba bean; however, the method for chickpea was not successful. Abstract Agroinfiltration is a valuable research method for investigating virulence and avirulence effector proteins from pathogens and pests, where heterologous effector proteins are transiently expressed in plant leaves and hypersensitive necrosis responses and other effector functions can be assessed. Nicotiana benthamiana is widely used for agroinfiltration and the characterisation of broad-spectrum effectors. The method has also been used in other plant species including field pea, but not yet developed for chickpea, lentil, or faba bean. Here, we have modified the pEAQ-HT-DEST1 vector for expression of 6 × histidine-tagged green-fluorescent protein (GFP) and the known necrosis-inducing broad-spectrum effector necrosis and ethylene-inducing peptide (Nep1)-like protein (NLP). Modified pEAQ-based vectors were adapted to encode signal peptide sequences for apoplast targeting of expressed proteins. We used confocal microscopy to assess the level of GFP expression in agroinfiltrated leaves. While at 3 days after infiltration in N. benthamiana, GFP was expressed at a relatively high level, expression in field pea and faba bean at the same time point was relatively low. In lentil, an expression level of GFP similar to field pea and faba bean at 3 days was only observed after 5 days. Chickpea leaf cells were transformed at low frequency and agroinfiltration was concluded to not be successful for chickpea. We concluded that the pEAQ vector is suitable for testing host-specific effectors in field pea, lentil, and faba bean, but low transformation efficiency limits the utility of the method for chickpea.

Integrative Conjugative Elements (ICEs) are replicons that can insert and excise from chromosomal locations in a site-specific manner, can conjugate across strains, and which often carry a variety of genes useful for bacterial growth and survival under specific conditions. Although ICEs have been identified and vetted within certain clades of the agricultural pathogen Pseudomonas syringae, the impact of ICE carriage and transfer across the entire P. syringae species complex remains underexplored. Here we identify and vet an ICE (PmaICE-DQ) from P. syringae pv. maculicola ES4326, a strain commonly used for laboratory virulence experiments, demonstrate that this element can excise and conjugate across strains, and highlight that this element contains loci encoding multiple type III effector proteins. Moreover, genome context suggests that another ICE (PmaICE-AOAB) is highly similar in comparison with and found immediately adjacent to PmaICE-DQ within the chromosome of strain ES4326, and also contains multiple type III effectors. Lastly, we present passage data from in planta experiments that suggests that genomic plasticity associated with ICEs may enable strains to more rapidly lose type III effectors that trigger R-gene mediated resistance in comparison to strains where nearly isogenic effectors are not present in active ICEs. Taken together, our study sheds light on a set of ICE elements from P. syringae pv. maculicola ES4326 and suggests how genomic context may lead to different evolutionary dynamics for shared virulence genes between strains.

Copper radical alcohol oxidases (CRO-AlcOx), which have been recently discovered among fungal phytopathogens, are attractive for the production of fragrant fatty aldehydes. With the initial objective to investigate the secretion of CRO-AlcOx by natural fungal strains, we undertook time course analyses of the secretomes of three Colletotrichum species (C. graminicola, C. tabacum, and C. destructivum) using proteomics. The addition of a copper-manganese-ethanol mixture in the absence of any plant-biomass mimicking compounds to Colletotrichum cultures unexpectedly induced the secretion of up to 400 proteins, 29 to 52% of which were carbohydrate-active enzymes (CAZymes), including a wide diversity of copper-containing oxidoreductases from the auxiliary activities (AA) class (AA1, AA3, AA5, AA7, AA9, AA11, AA12, AA13, and AA16). Under these specific conditions, while a CRO-glyoxal oxidase from the AA5_1 subfamily was among the most abundantly secreted proteins, the targeted AA5_2 CRO-AlcOx were secreted at lower levels, suggesting heterologous expression as a more promising strategy for CRO-AlcOx production and utilization. C. tabacum and C. destructivum CRO-AlcOx were thus expressed in Pichia pastoris, and their preference toward both aromatic and aliphatic primary alcohols was assessed. The CRO-AlcOx from C. destructivum was further investigated in applied settings, revealing a full conversion of C(6) and C(8) alcohols into their corresponding fragrant aldehydes. IMPORTANCE In the context of the industrial shift toward greener processes, the biocatalytic production of aldehydes is of utmost interest owing to their importance for their use as flavor and fragrance ingredients. Copper radical alcohol oxidases (CRO-AlcOx) have the potential to become platform enzymes for the oxidation of alcohols to aldehydes. However, the secretion of CRO-AlcOx by natural fungal strains has never been explored, while the use of crude fungal secretomes is an appealing approach for industrial applications to alleviate various costs pertaining to biocatalyst production. While investigating this primary objective, the secretomics studies revealed unexpected results showing that under the oxidative stress conditions we probed, Colletotrichum species can secrete a broad diversity of copper-containing enzymes (laccases, sugar oxidoreductases, and lytic polysaccharide monooxygenases [LPMOs]) usually assigned to “plant cell wall degradation,” despite the absence of any plant-biomass mimicking compound. However, in these conditions, only small amounts of CRO-AlcOx were secreted, pointing out recombinant expression as the most promising path for their biocatalytic application.

The genus Dickeya comprises plant pathogens that cause diseases in a large range of economically important crops and ornamentals. Strains previously assigned to the species Dickeya zeae are major pathogens attacking vital crops such as maize and rice. They are also frequently isolated from surface water. The newly described species Dickeya oryzae is closely related to D. zeae members, so that the limit between the two species can be difficult to define. In order to clearly distinguish the two species, globally described by the term ‘ D. zeae complex’, we sequenced the genome of four new water isolates and compared them to 14 genomes available in databases. Calculation of average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values confirmed the phylogenomic classification into the two species D. zeae and D. oryzae . It also allowed us to propose a new species, Dickeya parazeae sp. nov., to characterize a clade distinct from those containing the D. zeae type strain NCPPB2538T. Strain S31T (CFBP 8716T=LMG 32070T) isolated from water in France is proposed as the type strain of the new species. Phenotypic analysis of eight publically available strains revealed traits common to the five tested D. oryzae members but apparently not shared by the D. oryzae type strain. Genomic analyses indicated that a simple distinction between the species D. zeae , D. parazeae and D. oryzae can be obtained on the basis of the recA sequence. D. oryzae can be distinguished from the two other species by growth on l-tartaric acid. Based on the recA marker, several strains previously identified as D. zeae were re-assigned to the species D. parazeae or D. oryzae . This study also highlighted the broad host range diversity of these three species.

Endophytic microorganisms are found within the tissues of many plants species, with some conferring several benefits to the host plant including resistance to plant diseases. In this study, two putative endophytic fungi that were previously isolated from wild seeds of Brassica, identified as Beauveria bassiana and Pseudogymnoascus pannorum, were inoculated into cultivars of three Brassica species—Brassica napus, Br. rapa and Br. oleracea. Both fungal endophytes were reisolated from above- and below-ground tissues of inoculated plants at four different plant-growth stages, including cotyledon, one-leaf, two-leaf, and four-leaf stages. None of the plants colonised by these fungi exhibited any obvious disease symptoms, indicating the formation of novel mutualistic associations. These novel plant–endophyte associations formed between Brassica plants and Be. bassiana significantly inhibited phoma stem canker, a devastating disease of Brassica crops worldwide, caused by the fungal pathogen Leptosphaeria maculans. The novel association formed with P. pannorum significantly suppressed the amount of disease caused by L. maculans in one out of two experiments. Although biological control is not a new strategy, endophytic fungi with both antiinsect and antifungal activity are a highly conceivable, sustainable option to manage pests and diseases of economically important crops.

To discover microorganisms that naturally possess chemical weapons against the phytopathogen Fusarium oxysporum, the biological and chemical diversity of plant leaf endophytes was investigated. Endophytes were isolated from the palm tree Astrocaryum sciophyllum collected in pristine forests of French Guiana. Several Xylariaceae inhibited the growth of F. oxysporum and were further explored. Antifungal specialized metabolites were isolated from the Xylariaceae BSNB-0294 strain in confrontation with the phytopathogen and led to the identification of undescribed compounds, i.e., two depsipeptides named xylariaceins, two metabolites containing a 3-imidazolinone moiety, and four new compounds including a nitro-phenylpropanamide and three phenylalanine analogues named xylariains A-D. In parallel, the chemical investigation of the phytopathogen during the coculture led to the identification of an unknown compound, which we named focicin. The production of focicin was exacerbated during the competition. Matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectometry (MALDI-TOF MS) imaging of the competition between BSNB-0294 (endophytic strain) and F. oxysporum f.sp. ciceris (phytopathogen) highlighted time-dependent chemical interactions between the two microorganisms.

One of the relevant problems in today's agriculture is related to phytopathogenic microorganisms that cause between 30–40% of crop losses. Synthetic chemical pesticides and antibiotics have brought human and environmental health problems and microbial resistance to these treatments. So, the search for natural alternatives is necessary. The genus Streptomyces have broad biotechnological potential, being a promising candidate for the biocontrol of phytopathogenic microorganisms. The efficacy of some species of this genus in plant protection and their continued presence in the intensely competitive rhizosphere is due to its great potential to produce a wide variety of soluble bioactive secondary metabolites and volatile organic compounds. However, more attention is still needed to develop novel formulations that could increase the shelf life of streptomycetes, ensuring their efficacy as a microbial pesticide. In this sense, encapsulation offers an advantageous and environmentally friendly option. The present review aims to describe some phytopathogenic microorganisms with economic importance that require biological control. In addition, it focuses mainly on the Streptomyces genus as a great producer of secondary metabolites that act on other microorganisms and plants, exercising its role as biological control. The review also covers some strategies and products based on Streptomyces and the problems of its application in the field.

Trials are currently being conducted in South Africa to establish Amaranthus cruentus as a new pseudocereal crop. During recent surveys, Fusarium species were associated with weevil damage in A. cruentus fields. Preliminary studies showed that some of these Fusarium species grouped into two distinct clades within the F. fujikuroi species complex. The aim of this study was to characterize these isolates based on the morphology and phylogeny of the translation elongation factor 1α (TEF1α) gene region, ß-tubulin 2 (ßT) gene region and RNA polymerase II subunit (RPB2), and to determine if these isolates are pathogenic to A. cruentus. Phylogenetic and morphological studies showed that these two clades represent two novel species described here as F. casha and F. curculicola. Both species were shown to have the potential to be pathogenic to A. cruentus during routine greenhouse inoculation tests. While isolations indicate a possible association between these two species and weevils, further research is needed to understand this association and the role of weevils in disease development involving F. casha and F. curculicola in A. cruentus.

Fusarium verticillioides is the most predominant fungal phytopathogen of cereals and it is posing great concern from a global perspective. The fungus is mainly associated with maize, rice, sorghum, wheat, sugarcane, banana, and asparagus and causes cob, stalk, ear, root, crown, top, and foot rot. F. verticillioides produces fumonisins as the major secondary metabolite along with trace levels of beauvericin, fusaric acid, fusarin C, gibberiliformin, and moniliformin. Being a potential carcinogen, fumonisins continue to receive major attention as they are common contaminants in cereals and its processed food products. The importance of elimination of F. verticillioides growth and its associated fumonisin from cereals cannot be overemphasized considering the significant health hazards associated with its consumption. Physical and chemical approaches have been shown to reduce fumonisin B1 concentrations among feeds and food products but have proved to be ineffective during the production process. Hence, biological control methods using microorganisms, plant extracts, antioxidants, essential oils, phenolic compounds, and other advanced technologies such as growing disease-resistant crops by applying genetic engineering, have become an effective alternative for managing F. verticillioides and its toxin. The different methods, challenges, and concerns regarding the biocontrol of F. verticillioides and production of fumonisin B1 have been addressed in the present review.

Background Plant pathogenesis related-1 (PR-1) proteins belong to the CAP superfamily and have been characterized as markers of induced defense against pathogens. Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that respectively cause the witches’ broom disease and frosty pod rot in Theobroma cacao. Interestingly, a large number of plant PR-1-like genes are present in the genomes of both species and many are up-regulated during the biotrophic interaction. In this study, we investigated the evolution of PR-1 proteins from 22 genomes of Moniliophthora isolates and 16 other Agaricales species, performing genomic investigation, phylogenetic reconstruction, positive selection search and gene expression analysis. Results Phylogenetic analysis revealed conserved PR-1 genes (PR-1a, b, d, j), shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora (PR-1f, g, h, i), as well as in recent specialization cases within M. perniciosa biotypes (PR-1c, k, l) and M. roreri (PR-1n). PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host–pathogen arms race. Furthermore, although previous work showed that MpPR-1 can detoxify plant antifungal compounds in yeast, we found that in the presence of eugenol M. perniciosa differentially expresses only MpPR-1e, k, d, of which two are not linked to pathogenicity, suggesting that detoxification might not be the main function of most MpPR-1. Conclusions Based on analyses of genomic and expression data, we provided evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Additionally, we also discuss how fungal PR-1 proteins could have adapted from basal conserved functions to possible roles in fungal pathogenesis.

We report here the complete genome sequence of the Rhizobium rhizogenes (formerly Agrobacterium rhizogenes) strain LBA9402 (NCPPB1855rifR), a pathogenic strain causing hairy root disease. To assemble a complete genome, we obtained short reads from Illumina sequencing and long reads from Oxford Nanopore Technology sequencing. The genome consists of a 3,958,212 bp chromosome, a 2,005,144 bp chromid (secondary chromosome) and a 252,168 bp Ri plasmid (pRi1855), respectively. The primary chromosome was very similar to that of the avirulent biocontrol strain K84, but the chromid showed a 724 kbp deletion accompanied by a large 1.8 Mbp inversion revealing the dynamic nature of these secondary chromosomes. The sequence of the agropine Ri plasmid was compared to other types of Ri and Ti plasmids. Thus, we identified the genes responsible for agropine catabolism, but also a unique segment adjacent to the TL region that has the signature of a new opine catabolic gene cluster including the three genes that encode the three subunits of an opine dehydrogenase. Our sequence analysis also revealed a novel gene at the very right end of the TL‐DNA, which is unique for the agropine Ri plasmid. The protein encoded by this gene was most related to the succinamopine synthases of chrysopine and agropine Ti plasmids and thus may be involved in the synthesis of the unknown opine that can be degraded by the adjacent catabolic cluster. The available sequence will facilitate the use of R. rhizogenes and especially LBA9402 in both the laboratory and for biotechnological purposes., Comparative analysis revealed that the primary chromosome in Rhizobium rhizogenes was strongly conserved but the secondary chromosome was highly dynamic, showing huge rearrangements including both a large deletion and inversion. Detailed analysis of the agropine Ri plasmid suggested the evolution of a novel opine system. We found in the TL region a gene encoding a putative novel opine synthase that is evolutionarily related to the succinamopine synthases from agropine and chrysopine Ti plasmids. Also, in an adjacent unique region of the plasmid, we identified three genes encoding proteins with the signature of constituting the three subunits of an opine dehydrogenase.

The study was carried out to investigate the effect of soil mediated genetic change on growth rate of bacterial isolates from soils collected from different environments. Soil samples were collected from different sites: cow dung contaminated area, poultry, mechanic workshop, river, farmland and garden soils. Physiochemical analysis, bacteria isolation and enumeration were carried out on the different soil samples. Growth rate was determined by the measurement of turbidity at 600 nm using UV/VIS spectrophotometer on all isolates from two selected species (Pseudomonas and Staphylococcus) because of their even distribution in the soil samples. Plasmid curing was carried out on the selected isolates using 1 ml of 10% Sodium Dodecyl Sulphate as the curing agent and the growth rate of the plasmid-cured isolates were investigated. The results obtained indicated that the soil samples varied in their physiochemical properties. There were differences in the growth rates of bacterial isolates from the different soils for both Pseudomonas and Staphylococcus spp. a comparison between growth rate of test bacteria isolates before and after plasmid curing also suggested that curing significantly affected the growth rate of the test organisms in some soil samples. The results from this study suggested that the soil environments from which the various bacterial isolates were obtained had effect on the genetics of the bacteria isolates. The future direction to this study is to perform a molecular profiling of the various isolates to determine species specificity and also test for their virulence.

Fungi are the primary infectious agents in plants causing significant economic losses in agroindustry. Traditionally, these pathogens have been treated with different synthetic fungicides such as hydroxianilides, anilinopyrimidines, and azoles, to name a few. However, the indiscriminate use of these chemicals has increased fungi resistance in plants. Natural products have been researched as a control, and an alternative to these synthetic fungicides since they are not harmful to health and contribute to the environment caring. This review describes plants extracts, essential oils, and active compounds or secondary metabolites as antifungal agents both, in vitro and in vivo. Active compounds have been recently described as the best candidates for the control of phytopathogenic fungi. When metabolized by plants, these compounds concentrations rely on the environmental conditions and pathogens incidence. However, one issue regarding the direct application of these preformed compounds in plants touch upon their low persistence in the environment, and their even lower bioavailability than synthetic fungicides. Hence the challenge is to develop useful formulations based on natural products to increase the compounds solubility facilitating thus their application in the field while maintaining their properties. © 2019 Sociedad Chilena de Quimica. All rights reserved.

Massive use of wood biomass is usually associated with its long-term, large-scale storage in power plants and heating plants. Long-term storage of wood biomass (more than 3 months), in large volumes, brings risks from the point of view of human health or property treatment. This work aimed to verify how the long-term storage of wood chips from beech wood in small piles affects their energy properties and whether in this way it is possible to reduce the risk of fire by self-heating in piles and the volume of phytopathogenic spore production. Four experimental piles, each with a base of 4 × 4 m and a height of 2 m, were established. After 6 months, one of the piles was disassembled and samples from 0.5 m, 1.0 m, and 1.5 m height levels were taken for analysis. The results of the experiment confirmed that the energy properties of wood chips stored in small piles significantly deteriorate after more than half a year of storage. It has also been confirmed that the choice of this method of storing in smaller, spatially divided piles can lead to a significant minimization of the risk of spontaneous combustion and fire. The length of the storage period did not have a positive effect on the abundance of phytopathogen content in the stored piles. With the length of storage, only the number of identified harmful species of phytopathogens were changed and, at the same time, their number of colonies increased.

Inonotus rickii(Pat.) Reid (Agaricomycetes: Hymenochaetaceae) is a poroid fungus characterized by the expressive production of chlamydospores,in vivoandin vitro, especially during its anamorphic stage. The species plays important ecological roles, standing out as a phytopathogen, affecting several species of ornamental and wild trees, mainly in tropical and subtropical regions. The infected trees develop canker and white rot of the wood, showing symptoms of reduced vegetative vigor and decline of leaves and branches which causes death in some cases. The first record ofI. rickiifor the Cerrado biome (Brazilian Savanna) and the first record as causal agent of canker inSchinus molleL. in Brazil is reported here. In addition, we present a checklist of its worldwide geographical distribution and known hosts, from an extensive bibliographic search in Google Scholar, SciELO, Scopus, and Web of Science databases. The species is widespread in tropical and subtropical zones; common in the American continent, especially in Central and South America and the Mediterranean region, and rare in temperate zones. We found specimens growing in both living and dead hosts, totalizing 70 species of hosts, distributed in 43 genera and 22 families. Of these,Acer negundoL. (10.5%),Celtis australisL. (6.5%), andPlatanus acerifolia(Aiton) Willd. (4.8%), and the Fabaceae (30%), Fagaceae (10%), and Sapindaceae (8.6%) families were the most frequent. We present morphological descriptions and illustrations, as well as the growth characteristics in culture medium. Our study expands the known geographical distribution ofI. rickii, including the Cerrado biome, as well as its structural, physiological characteristics, and its hosts.

Citrus canker disease, caused by the bacterium Xanthomonas citri subsp. citri is a constant threat to citrus-producing areas. Since it has no cure, agricultural practices to restrain its dissemination are essential to reduce the economic damage. Hence, increased knowledge of the basic aspects of X. citri biology could lead to more efficient management practices that can eliminate dormant bacteria in the field. The dormant cells, also referred to as persisters, are phenotypic variants with lowered metabolism, which in turn leads to tolerance to antimicrobials and undermines existing control approaches. We show here that X. citri forms persisters, identifying triggers for this phenotype, including antibiotics, high temperature, and metals (copper and zinc), which increase persistence rates by 10–100 times. The antioxidant N-acetylcysteine reduced copper and zinc-induced persisters, but not those induced by tetracycline, indicating that oxidative stress may be an important inducer of X. citri persistence. In addition, we found that metabolism-independent drugs like cisplatin and mitomycin C are able to eliminate X. citri persistent cells, as well as copper, at high concentrations. Specific amino acids like proline and isoleucine interfered with the physiological balance of the dormancy in X. citri, stimulating or preventing persister resuscitation. Taken together, we discover chemicals that can induce, wake, and kill X. citri persister cells, these results provide insights that should be considered for more efficient integrated control management in the field.

Background Microorganisms, including Bacillus species are used to help control plant pathogens, thereby reducing reliance on synthetic pesticides in agriculture. Bacillus velezensis strain 1B-23 has been shown to reduce symptoms of bacterial disease caused by Clavibacter michiganensis subsp. michiganensis in greenhouse-grown tomatoes, with in vitro studies implicating the lipopeptide surfactin as a key antimicrobial. While surfactin is known to be effective against many bacterial pathogens, it is inhibitory to a smaller proportion of fungi which nonetheless cause the majority of crop diseases. In addition, knowledge of optimal conditions for surfactin production in B. velezensis is lacking. Results Here, B. velezensis 1B-23 was shown to inhibit in vitro growth of 10 fungal strains including Candida albicans, Cochliobolus carbonum, Cryptococcus neoformans, Cylindrocarpon destructans Fusarium oxysporum, Fusarium solani, Monilinia fructicola, and Rhizoctonia solani, as well as two strains of C. michiganensis michiganensis. Three of the fungal strains (C. carbonum, C. neoformans, and M. fructicola) and the bacterial strains were also inhibited by purified surfactin (surfactin C, or [Leu7] surfactin C15) from B. velezensis 1B-23. Optimal surfactin production occurred in vitro at a relatively low temperature (16 °C) and a slightly acidic pH of 6.0. In addition to surfactin, B. velenzensis also produced macrolactins, cyclic dipeptides and minor amounts of iturins which could be responsible for the bioactivity against fungal strains which were not inhibited by purified surfactin C. Conclusions Our study indicates that B. velezensis 1B-23 has potential as a biocontrol agent against both bacterial and fungal pathogens, and may be particularly useful in slightly acidic soils of cooler climates.

Some Plant Growth-Promoting Rhizobacteria (PGPR) can induce protection against pathogens, increasing plant tolerance to various diseases. This so-called biocontrol activity is replacing harmful practices in agriculture caused by the use of agrochemicals. Azospirillum brasilense is one of the PGPR already effectively used as a resistance inducer in several crops. The aim of this study was to evaluate the protective effect of PGPR A. brasilense strains isolated from strawberry and petunia plants (REC3, 2A1, 2A2, and 2E1) against the fungal pathogen Macrophomina phaseolina, which is the causal agent of the strawberry charcoal rot disease. In vitro antagonism assays and enzymatic tests on Petri dishes revealed no direct inhibition on M. phaseolina growth by any of the A. brasilense strains. However, strawberry plants treated with REC3 and 2A1 strains increased callose and lignin deposition and stomatal closure compared to untreated plants. In addition, treatments with either bacterial strains induced a defense response in strawberry plants against virulent isolates of M. phaseolina evidenced by an increased tolerance to the charcoal rot disease. These results suggest that A. brasilense REC3 and 2A1 strains can be used for the activation of innate immunity in strawberry plants as a strategy for managing charcoal rot in a sustainable and environmentally friendly way. EEA Famaillá Fil: Viejobueno, Josefina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Famaillá; Argentina Fil: Albornoz, Patricia Liliana. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales; Argentina Fil: Albornoz, Patricia Liliana. Fundación Miguel Lillo; Argentina Fil: Camacho, María. Instituto de Investigación y Formación Agraria y Pesquera; España Fil: Santos, Berta de los. Instituto de Investigación y Formación Agraria y Pesquera; España Fil: Martinez Zamora, Martin Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Martinez Zamora, Martin Gustavo. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Salazar, Sergio Miguel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Famaillá; Argentina Fil: Salazar, Sergio Miguel. Universidad Nacional de Tucumán. Facultad de Agronomía y Zootecnia; Argentina

1. Pine wilt disease, caused by pinewood nematode (Bursaphelenchus xylophilus; abbreviated ‘PWN’), is a damaging and globally distributed insect‐vectored forest pathogen. Native forest tree mortality associated with PWN is newly reported from the Front Range of Colorado, but there is no regional information on PWN frequency or biology of local insect vectors, limiting management options. 2. A sampling array was established to survey PWN in native pines (Pinus ponderosa) and longhorn beetles (Monochamus clamator and M. scutellatus) over 2 years and across natural and urban forest landscapes. We developed flight phenology models and evaluated effects of landscape factors on vector abundance and probability of infection. 3. Flight phenology was similar for vectors; Monochamus flight initiated in mid‐July and continued into October for both species. We report the first M. clamator–PWN association in the United States. PWN was distributed in the region at rates lower than reported from its putative native range: 3.6 and 4.2% of sampled pines and beetles, respectively, tested positive for PWN. Many host trees were outwardly asymptomatic; infection frequency in tree populations varied considerably and four epicentres of vector infectivity were identified. 4. Epicentres varied in timing of anomalous infective vector frequency – some epicentres had high abundances of infected beetles early in the growing season whereas others had high abundances of infected beetles late in the growing season, though PWN‐positive beetles were captured at all sites. Monochamus populations were found primarily in natural forest stands but migrated to urban areas late in the growing season. The only landscape factor positively correlated with abundances of both Monochamus species was distance to previous wildfire. 5. PWN epicentres in the southern Rocky Mountains exhibit specific temporal windows of vector activity that differ from proximal sites. Urban forests, where the disease was initially observed in the region, do not support vector populations. Our results suggest that natural forest landscapes in the region are important reservoirs of PWN, and vector populations are especially abundant near burned stands. Collectively, our findings are important for timing disease management activities appropriately and help to distinguish priority areas for mitigation efforts.

The genus Neofusicoccum is comprised of approximately 50 species with a worldwide distribution and is typically associated with plants. Neofusicoccum is well-known for the diseases it causes on economically and ecologically relevant host plants. In particular, members of this genus are responsible for grapevine diseases, such as leaf spots, fruit rots, shoot dieback, bud necrosis, vascular discoloration of the wood, and perennial cankers. Many secondary metabolites, including (−)-botryoisocoumarin A, botryosphaerones, cyclobotryoxide and isosclerone, were identified from species of Neofusicoccum and their structural variability and bioactivities might be associated with the role of these compounds in the fungal pathogenicity and virulence. In this review, we summarize the secondary metabolites from Neofusicoccum species focusing on the role of these compounds in the interaction between the fungus and host plant.

In Australia, Stylosanthes little leaf (StLL) phytoplasma has been detected in Stylosanthes scabra Vogel, Arachis pintoi Krapov, Saccharum officinarum L., Carica papaya L., Medicago sativa L., and Solanum tuberosum L. The 16S rRNA gene sequence of StLL phytoplasma strains from S. scabra, C. papaya, S. officinarum and S. tuberosum were compared and share 99.93–100 % nucleotide sequence identity. Phylogenetic comparisons between the 16S rRNA genes of StLL phytoplasma and other ‘Candidatus Phytoplasma’ species indicate that StLL represents a distinct phytoplasma lineage. It shares its most recent known ancestry with ‘Ca. Phytoplasma luffae’ (16SrVIII-A), with which it has 97.17–97.25 % nucleotide identity. In silico RFLP analysis of the 16S rRNA amplicon using iPhyClassifier indicate that StLL phytoplasmas have a unique pattern (similarity coefficient below 0.85) that is most similar to that of ‘Ca. Phytoplasma luffae’. The unique in silico RFLP patterns were confirmed in vitro. Nucleotide sequences of genes that are more variable than the 16S rRNA gene, namely tuf (tu-elongation factor), secA (partial translocation gene), and the partial ribosomal protein (rp) gene operon (rps19-rpl22-rps3), produced phylogenetic trees with similar branching patterns to the 16S rRNA gene tree. Sequence comparisons between the StLL 16S rRNA spacer region confirmed previous reports of rrn interoperon sequence heterogeneity for StLL, where the spacer region of rrnB encodes a complete tRNA-Isoleucine gene and the rrnA spacer region does not. Together these results suggest that the Australian phytoplasma, StLL, is unique according to the International Organization for Mycoplasmology (IRPCM) recommendations. The novel taxon ‘Ca. Phytoplasma stylosanthis’ is proposed, with the most recent strain from a potato crop in Victoria, Australia, serving as the reference strain (deposited in the Victorian Plant Pathology Herbarium as VPRI 43683).

In recent years, there has been an increase in the number of diseases caused by bacterial, fungal, and viral infections. Infections affect plants at different stages of agricultural production. Depending on weather conditions and the phytosanitary condition of crops, the prevalence of diseases can reach 7080% of the total plant population, and the yield can decrease in some cases down to 8098%. Plants have innate cellular immunity, but specific phytopathogens have an ability to evade that immunity. This article examined phytopathogens of viral, fungal, and bacterial nature and explored the concepts of modern plant protection, methods of chemical, biological, and agrotechnical control, as well as modern methods used for identifying phytopathogens.

Xanthomonas euvesicatoria (Xe) is a gram-negative phytopathogenic bacterium that causes bacterial spot disease in tomato/pepper leading to economic losses in plantations. DNA methyltransferases (MTases) are critical for the survival of prokaryotes; however, their functions in phytopathogenic bacteria remain unclear. In this study, we characterized the functions of two putative DNA MTases, XvDMT1 and XvDMT2, in Xe by generating XvDMT1- and XvDMT2-overexpressing strains, Xe(XvDMT1) and Xe(XvDMT2), respectively. Virulence of Xe(XvDMT2), but not Xe(XvDMT1), on tomato was dramatically reduced. To postulate the biological processes involving XvDMTs, we performed a label-free shotgun comparative proteomic analysis, and results suggest that XvDMT1 and XvDMT2 have distinct roles in Xe. We further characterized the functions of XvDMTs using diverse phenotypic assays. Notably, both Xe(XvDMT1) and Xe(XvDMT2) showed growth retardation in the presence of sucrose and fructose as the sole carbon source, with Xe(XvDMT2) being the most severely affected. In addition, biofilm formation and production of exopolysaccharides were declined in Xe(XvDMT2), but not Xe(XvDMT1). Xe(XvDMT2) was more tolerant to EtOH than Xe(XvDMT1), which had enhanced tolerance to sorbitol but decreased tolerance to polymyxin B. Using single-molecule real-time sequencing and methylation-sensitive restriction enzymes, we successfully predicted putative motifs methylated by XvDMT1 and XvDMT2, which are previously uncharacterized 6mA and 5mC DNA MTases, respectively. This study provided new insights into the biological functions of DNA MTases in prokaryotic organisms.

Made available in DSpace on 2020-12-12T02:23:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-09-02 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Universidade Federal de Uberlândia Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Xylella fastidiosa colonizes the xylem of various cultivated and native plants worldwide. Citrus production in Brazil has been seriously affected, and major commercial varieties remain susceptible to Citrus Variegated Chlorosis (CVC). Collective cellular behaviors such as biofilm formation influence virulence and insect transmission of X. fastidiosa. The reference strain 9a5c produces a robust biofilm compared to Fb7 that remains mostly planktonic, and both were isolated from symptomatic citrus trees. This work deepens our understanding of these distinct behaviors at the molecular level, by comparing the cellular and secreted proteomes of these two CVC strains. Out of 1017 identified proteins, 128 showed differential abundance between the two strains. Different protein families were represented such as proteases, hemolysin-like proteins, and lipase/esterases, among others. Here we show that the lipase/esterase LesA is among the most abundant secreted proteins of CVC strains as well, and demonstrate its functionality by complementary activity assays. More severe symptoms were observed in Nicotiana tabacum inoculated with strain Fb7 compared to 9a5c. Our results support that systemic symptom development can be accelerated by strains that invest less in biofilm formation and more in plant colonization. This has potential application in modulating the bacterial-plant interaction and reducing disease severity. Institute of Biotechnology Federal University of Uberlandia, Av. Amazonas, Bloco 2E, Campus Umuarama Department of Plant Sciences College of Agriculture and Environmental Sciences University of California Davis, 1 Shields Ave Department of Technology School of Agricultural and Veterinary Studies São Paulo State University (FCAV/UNESP), Via de Acesso Prof. Paulo Donato Castellane Citriculture Defense Fund (Fundecitrus), Av. Dr. Adhemar Pereira de Barros 201 Department of Biochemistry Institute of Chemistry University of São Paulo, Av. Prof. Lineu Prestes 748 Department of Technology School of Agricultural and Veterinary Studies São Paulo State University (FCAV/UNESP), Via de Acesso Prof. Paulo Donato Castellane FAPESP: 11/24091-0 FAPEMIG: 88881.064973/2014-01

Pectobacterium atrosepticum (Pba) is a quarantine and threatening phytopathogen known as the causal agent of blackleg and soft rot disease of potatoes in many areas. Its early detection is then important to have healthy potato tubers and reduce economic losses. Today, conventional methods such as enzyme-linked immunosorbent-assay (ELISA) and polymerase chain reaction (PCR) are typically used for Pba detection, but they are expensive and time-consuming. Here we report on the optimization of an alternative approach based on an electrochemical impedance immunosensor combining a microfluidic module and a microelectrodes array, and having advantages in terms of low cost, ease of use and portability. For validation and for assessing its performance, the lab-on-chip platform has been compared with two standard methods (ELISA and PCR).

Pseudomonas syringae is a plant pathogen, which groups over 40 pathovars. Climate change and international trade facilitate the worldwide spread of pathogenic P. syringae strains. In recent decades, infections with P. syringae have been causing large losses in vegetable growing and horticulture. With the aim to look for biocontrol agents that could minimize these losses, we isolated bacteriophages infective for certain P. syringae strains. One of these phages, designated by us as vB_PsyP_3MF5 (3MF5), appeared to have atypical properties. It formed clear plaques on the layers of sensitive cells at elevated temperatures but was unable to form plaques at room temperature. It quickly adsorbed to its host cells and had a short latent period and a large burst size at permissive temperature. However, several survivors of the phage infection could be isolated in a standard killing assay. They appeared to form a lysis zone when placed on a layer of cells that were not treated with this phage, indicating that they are 3MF5 lysogens. In support of that, their DNA could serve as a template for PCR amplification with 3MF5 specific primer pairs. The analysis of the 3MF5 genomic sequence (GenBank. Acc. No. MT597419) revealed features typical of Teseptimavirus genus phages which are obligatorily lytic and are unable to lysogenize cells. Additionally, comparative analysis of the predicted 3MF5 proteins excluded the presence of any obvious homolog of a typical phage repressor that inhibits transcription of early phage genes in lysogens. Conceivably, the repression is achieved either by the interaction of a temperature-sensitive host/phage protein with a region controlling the expression of phage early genes or by temperature-induced structural changes in phage RNA, which could act by the occlusion of ribosomal binding sites of early phage genes. Surprisingly, the results of our preliminary studies indicate that despite its conditionally temperate nature, 3MF5 exhibits biocontrol properties.

To date, Certrevirus is one of two genera of bacteriophage (phage), with phages infecting Pectobacterium atrosepticum, an economically important phytopathogen that causes potato blackleg and soft rot disease. This study provides a detailed description of Pectobacterium phage CB7 (vB_PatM_CB7), which specifically infects P. atrosepticum. Host range, morphology, latent period, burst size and stability at different conditions of temperature and pH were examined. Analysis of its genome (142.8 kbp) shows that the phage forms a new species of Certrevirus, sharing sequence similarity with other members, highlighting conservation within the genus. Conserved elements include a putative early promoter like that of the Escherichia coli sigma70 promoter, which was found to be shared with other genus members. A number of dissimilarities were observed, relating to DNA methylation and nucleotide metabolism. Some members do not have homologues of a cytosine methylase and anaerobic nucleotide reductase subunits NrdD and NrdG, respectively. Furthermore, the genome of CB7 contains one of the largest numbers of homing endonucleases described in a single phage genome in the literature to date, with a total of 23 belonging to the HNH and LAGLIDADG families. Analysis by RT-PCR of the HNH homing endonuclease residing within introns of genes for the large terminase, DNA polymerase, ribonucleotide reductase subunits NrdA and NrdB show that they are splicing competent. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was also performed on the virion of CB7, allowing the identification of 26 structural proteins&mdash, 20 of which were found to be shared with the type phages of the genera of Vequintavirus and Seunavirus. The results of this study provide greater insights into the phages of the Certrevirus genus as well as the subfamily Vequintavirinae.

Chocolate spot is a major fungal disease of faba bean caused by the ascomycete fungus, Botrytis fabae. B. fabae is also implicated in botrytis gray mold disease in lentils, along with B. cinerea. Here we have isolated and characterized two B. fabae isolates from chocolate spot lesions on faba bean leaves. In plant disease assays on faba bean and lentil, B. fabae was more aggressive than B. cinerea and we observed variation in susceptibility among a small set of cultivars for both plant hosts. Using light microscopy, we observed a spreading, generalized necrosis response in faba bean toward B. fabae. In contrast, the plant response to B. cinerea was localized to epidermal cells underlying germinated spores and appressoria. In addition to the species characterization of B. fabae, we produced genome assemblies for both B. fabae isolates using Illumina sequencing. Genome sequencing coverage and assembly size for B. fabae isolates, were 27x and 45x, and 43.2 and 44.5 Mb, respectively. Following genome assembly and annotation, carbohydrate-active enzyme (CAZymes) and effector genes were predicted. There were no major differences in the numbers of each of the major classes of CAZymes. We predicted 29 effector genes for B. fabae, and using the same selection criteria for B. cinerea, we predicted 34 putative effector genes. For five of the predicted effector genes, the pairwise dN/dS ratio between orthologs from B. fabae and B. cinerea was greater than 1.0, suggesting positive selection and the potential evolution of molecular mechanisms for host specificity in B. fabae. Furthermore, a homology search of secondary metabolite clusters revealed the absence of the B. cinerea phytotoxin botrydial and several other uncharacterized secondary metabolite biosynthesis genes from B. fabae. Although there were no obvious differences in the number or proportional representation of different transposable element classes, the overall proportion of AT-rich DNA sequence in B. fabae was double that of B. cinerea.

Two homologous gene clusters for the biosynthesis of sesterterpenes betaestacins were identified from two phytopathogens, Phoma betae and Colletotrichum orbiculare. Heterologous expression of identified oxidation enzymes with previously-characterized PbTS1 (BtcA(pb)) resulted in the production of seven novel sesterterpenes. Although both strains possessed homologous enzymes, oxidation state of corresponding products were different from each other, suggesting that structural diversification of sesterterpene skeletons might be achieved by these homologous enzymes with different functions. (C) 2013 Elsevier Ltd. All rights reserved.

Potato is an important staple food crop. Potato tubers require proper treatment before planting and after harvest to produce high yields and avoid storage losses. Among different techniques of potato treatment physical methods are of special interest: thermal treatment using hot water and steam, ultraviolet (including continuous-wave UV using pulsed Xe-lamps) and gamma-irradiation, treatment with magnetic and electromagnetic fields (including microwaves). The majority of physical methods is environmentally friendly and can be applied without special registration and in the developing countries. In the present paper, for the first time, the scientific papers on physical methods of potato treatment for the last 35 years are comprehensively reviewed. The review demonstrates that such an approach is perspective both for pre-planting and postharvest treatment of potato. Physical treatment affects biochemical, cellular and physiological status of potato. Methods of physical treatment enable to control phytopathogens, and some methods (ultraviolet and gamma-radiation) even are capable of improving immunity of plants. The main traits of potato tubers that can be influenced by physical treatment are sprouting (stimulation or inhibition), susceptibility to rot and black leg diseases, and starch, reducing sugars and ascorbic acid contents. The tuber response to physical treatment depends on dosage and date of treatment, duration and temperature of storage, agricultural technology and cultivar. Low doses of treatment may be inefficient while too high dosage may result in cell deterioration or death and poor immunity, and eventually to disease development. Too early treatment may damage a tuber since it should pass through suberization (wound healing) after harvest; too late treatment requires higher doses. The proper adjustment of treatment is necessary for cultivar and individual storage conditions.

White mold disease, caused by fungus Sclerotinia sclerotiorum (Lib.) de Bary., is a disease hard to control due to the high amount of sclerotia produced, which guarantees its survival in the soil for years leading to significant yield losses. Alternative techniques to control the pathogen have been researched, including homeopathy. The present work aimed to evaluate the in vitro antifungal effect of homeopathic medicines on S. sclerotiorum mycelial growth. Homeopathic medicines Sulphur, fungal sclerotium Nosode and Calcarea carbonica, in 30CH, 200CH and 1000CH dynamizations were tested. Assays were carried out in a completely randomized design, with four repetitions. Experiments were performed through the addition of homeopathic medicines on the surface of plates containing culture medium, followed by insertion of a disc containing fungus mycelia and incubation. Control treatment received no homeopathic medicine. The mycelial progression was monitored by seven halo diameter measurements during experiment period. All homeopathic medicines tested and their dynamizations were able to inhibit partially the development of the fungus. Calcarea carbonica at the dynamization of 1000 CH showed the best inhibitory effect on S. sclerotiorum, which under its effect produced a mycelial halo 40% smaller than the control treatment. White mold disease, caused by fungus Sclerotinia sclerotiorum (Lib.) de Bary., is a disease hard to control due to the high amount of sclerotia produced, which guarantees its survival in the soil for years leading to significant yield losses. Alternative techniques to control the pathogen have been researched, including homeopathy. The present work aimed to evaluate the in vitro antifungal effect of homeopathic medicines on S. sclerotiorum mycelial growth. Homeopathic medicines Sulphur, fungal sclerotium Nosode and Calcarea carbonica, in 30CH, 200CH and 1000CH dynamizations were tested. Assays were carried out in a completely randomized design, with four repetitions. Experiments were performed through the addition of homeopathic medicines on the surface of plates containing culture medium, followed by insertion of a disc containing fungus mycelia and incubation. Control treatment received no homeopathic medicine. The mycelial progression was monitored by seven halo diameter measurements during experiment period. All homeopathic medicines tested and their dynamizations were able to inhibit partially the development of the fungus. Calcarea carbonica at the dynamization of 1000 CH showed the best inhibitory effect on S. sclerotiorum, which under its effect produced a mycelial halo 40% smaller than the control treatment.

Determination of the biocompatibility of microorganisms isolated from natural sources (Kemerovo Oblast—Kuzbass) resulted in the creation of three microbial consortia based on the isolated strains: consortium I (Bacillus pumilus, Pediococcus damnosus, and Pediococcus pentosaceus), consortium II (Acetobacter aceti, Pseudomonas chlororaphis, and Streptomyces parvus), and consortium III (Amycolatopsis sacchari, Bacillus stearothermophilus, Streptomyces thermocarboxydus, and Streptomyces thermospinisporus). The nutrient media composition for the cultivation of each of the three studied microbial consortia, providing the maximum increase in biomass, was selected: consortium I, nutrient medium 11, consortium II, nutrient medium 13, for consortium III, nutrient medium 16. Consortia I and II microorganisms were cultured at 5–25 °C, and consortium III at 50–70 °C. Six types of psychrophilic microorganisms (P. pentosaceus, P. chlororaphis, P. damnosus, B. pumilus, A. aceti, and S. parvus) and four types of thermophilic microorganisms (B. stearothermophilus, S. thermocarboxydus, S. thermospinisporus, and A. sacchari) were found to have high antagonistic activity against the tested pathogenic strains (A. faecalis, B. cinerea, E. carotovora, P. aeruginosa, P. fluorescens, R. stolonifera, X. vesicatoria. pv. Vesicatoria, and E. aphidicola). The introduction of microalgae hydrolyzate increased the concentration of microorganisms by 5.23 times in consortium I, by 4.66 times in consortium II, by 6.6 times in consortium III. These data confirmed the efficiency (feasibility) of introducing microalgae hydrolyzate into the biofertilizer composition.

Type II toxin-antitoxin (TA) systems are genetic elements usually encoding two proteins: a stable toxin and an antitoxin, which binds the toxin and neutralizes its toxic effect. The disturbance in the intracellular toxin and antitoxin ratio typically leads to inhibition of bacterial growth or bacterial cell death. Despite the fact that TA modules are widespread in bacteria and archaea, the biological role of these systems is ambiguous. Nevertheless, a number of studies suggests that the TA modules are engaged in such important processes as biofilm formation, stress response or virulence and maintenance of mobile genetic elements. The Dickeya dadantii 3937 strain serves as a model for pathogens causing the soft-rot disease in a wide range of angiosperm plants. Until now, several chromosome-encoded type II TA systems were identified in silico in the genome of this economically important bacterium, however so far only one of them was experimentally validated. In this study, we investigated three putative type II TA systems in D. dadantii 3937: ccdAB2Dda, phd-docDda and dhiTA, which represents a novel toxin/antitoxin superfamily. We provide an experimental proof for their functionality in vivo both in D. dadantii and Escherichia coli. Finally, we examined the prevalence of those systems across the Pectobacteriaceae family by a phylogenetic analysis.

Context: The Food and Agriculture Organization has estimated that every year considerable losses of the food crops occur due to plant diseases. Although fungicides are extensively used for management of plant diseases, they are expensive and hazardous to the environment and human health. Alternatively, biological control is the safe way to overcome the effects of plant diseases and to sustain agriculture. Since Monarda citriodora Cerv. ex Lag. (Lamiaceae/Labiatae) is known for its antifungal properties, it was chosen for the study. Objective: The isolation of endophytic fungi from M. citriodora and assessing their biocontrol potential. Material and methods: The isolated endophytes were characterized using ITS-5.8 S rDNA sequencing. Their biocontrol potential was assessed using different antagonistic assays against major plant pathogens. Results: Twenty-eight endophytes representing 11 genera were isolated, of which, around 82% endophytes showed biocontrol potential against plant pathogens. MC-2 L (Fusarium oxysporum), MC-14 F (F. oxysporum), MC-22 F (F. oxysporum) and MC-25 F (F. redolens) displayed significant antagonistic activity against all the tested pathogens. Interestingly, MC-10 L (Muscodor yucatanensis) completely inhibited the growth of Sclerotinia sp., Colletotrichum capsici, Aspergillus flavus and A. fumigatus in dual culture assay, whereas MC-8 L (A. oryzae) and MC-9 L (Penicillium commune) completely inhibited the growth of the Sclerotinia sp. in fumigation assay. Conclusions: Endophytes MC-2 L, MC-14 F, MC-22 F and MC-25 F could effectively be used to control broad range of phytopathogens, while MC-10 L, MC-8 L and MC-9 L could be used to control specific pathogens. Secondly, endophytes showing varying degrees of antagonism in different assays represented the chemo-diversity not only as promising biocontrol agents but also as a resource of defensive and bioactive metabolites.

Salmonella enterica is one of the most common pathogens associated with produce outbreaks worldwide; nonetheless, the mechanisms uncovering their interaction with plants are elusive. Previous reports demonstrate that S. enterica ser. Typhimurium (STm), similar to the phytopathogen Pseudomonas syringae pv. tomato (Pst) DC3000, triggers a transient stomatal closure suggesting its ability to overcome this plant defense and colonize the leaf apoplast. In order to discover new molecular players that function in the stomatal reopening by STm and Pst DC3000, we performed an Arabidopsis mutant screening using thermal imaging. Further stomatal bioassay confirmed that the mutant plants exo70h4-3, sce1-3, bbe8, stp1, and lsu2 have smaller stomatal aperture widths than the wild type Col-0 in response to STm 14028s. The mutants bbe8, stp1 and lsu2 have impaired stomatal movement in response to Pst DC3000. These findings indicate that EXO70H4 and SCE1 are involved in bacterial-specific responses, while BBE8, STP1, and LSU2 may be required for stomatal response to a broad range of bacteria. The identification of new molecular components of the guard cell movement induced by bacteria will enable a better understanding of the initial stages of plant colonization and facilitate targeted prevention of leaf contamination with harmful pathogens.

White grain disorder is a recently emerged wheat disease in Australia, caused by Eutiarosporella darliae, E. pseudodarliae, and E. tritici-australis. The disease cycle of these pathogens and the molecular basis of their interaction with wheat are poorly understood. To address this knowledge gap, we undertook a comparative genomics analysis focused on the secondary metabolite gene repertoire among these three species. This analysis revealed a diverse array of secondary metabolite gene clusters in these pathogens, including modular polyketide synthase genes. These genes have only been previously associated with bacteria and this is the first report of such genes in fungi. Subsequent phylogenetic analyses provided strong evidence that the modular PKS genes were horizontally acquired from a bacterial or a protist species. We also uncovered a secondary metabolite gene cluster with three polyketide/nonribosomal peptide synthase genes (Hybrid-1, -2, and -3) in E. darliae and E. pseudodarliae. In contrast, only remnant and partial genes homologous to this cluster were identified in E. tritici-australis, suggesting loss of this cluster. Homologues of Hybrid-2 in other fungi have been proposed to facilitate disease in woody plants, suggesting a possible alternative host range for E. darliae and E. pseudodarliae. Subsequent assays confirmed that E. darliae and E. pseudodarliae were both pathogenic on woody plants, but E. tritici-australis was not, implicating woody plants as potential host reservoirs for the fungi. Combined, these data have advanced our understanding of the lifestyle and potential host-range of these recently emerged wheat pathogens and shed new light on fungal secondary metabolism.

FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais Xylella fastidiosa é uma bactéria Gram-negativa e não flagelada responsável por colonizar o xilema de várias plantas cultivadas e selvagens encontradas em todo o mundo. Nas últimas décadas, tem sido bem estudada devido à importância deste fitopatógeno como agente causador de duas principais doenças, a Clorose Variegada dos Citros (CVC), no Brasil e na Argentina, e a Doença de Pierce (PD) nos Estados Unidos. Algumas cepas de X.fastidiosa têm seus genomas completamente ou parcialmente elucidados, como: 9a5c, isolado de laranja no estado de São Paulo e a cepa Fb7, isolada da laranja na região de Corrientes na Argentina. Baseando-se na análise genômica entre 9a5c e Fb7, e também na importância de uma lipase/esterase na patogenicidade de X.fastidiosa, o objetivo deste trabalho foi analisar e identificar os fatores associados à patogenicidade e virulência de X.fastidiosa 9a5c e Fb7. A fim de visualizar a presença de LesA nas amostras de proteínas totais e secretadas das cepas 9a5c e Fb7, testes esterásico e lipásico foram realizados. A atividade lipásica foi analisada cultivando a bactéria em meio de cultura suplementado com 1% de tributirina. Este teste demonstrou atividade sob os triacilgliceróis de cadeia curta presentes no meio e pode ser visualizado pela maior formação do halo na cepa Fb7. A atividade de esterase, determinada pelo substrato 4-metilumbelliferilbutirato (4-MUB), apresentou valores significativos e claramente expressos. Assim, a estirpe Fb7 apresentou atividade consideravelmente maior quando comparada com 9a5c. Em busca de outros fatores de virulência, foi realizado uma cromatografia líquida acoplada à espectrometria de massa (LC-MS/MS) das proteínas totais das culturas de X.fastidiosa de ambas as cepas. Nesta análise, foi possível identificar 119 proteínas diferencialmente expressas. Dentre elas, algumas proteínas com funções associadas a virulência da bactéria foram superexpressas na cepa Fb7, comparado a cepa 9a5c. A análise de RT-qPCR permitiu validar a expressão de algumas dessas proteínas. Os resultados demonstraram que a LesA e outras proteínas com níveis de expressão superior em Fb7, desempenham um importante papel de patogenicidade nesta cepa. Dessa forma, são fatores que podem explicar a maior virulência da cepa Fb7 quando comparado a cepa 9a5c. Xylella fastidiosa is a gram-negative and non-flagellate bacterium that colonizes the xylem of various cultivated and wild plants worldwide. In the last decades, it has been well studied due to the importance of this phytopathogen as the causing agent of two major diseases, Citrus Variegated Chlorosis (CVC) in Brazil and Argentina, and Pierce Disease (PD) in the United States. Some strains of X.fastidiosa have their genomes completely or partially elucidated, such as 9a5c, isolated from orange in the state of São Paulo, and the strain Fb7, isolated from orange in the region of Corrientes in Argentina. Based on the genomic analysis between strains 9a5c and Fb7, and on the importance of a lipase/esterase in the pathogenicity of X.fastidiosa, the objective of this work was to analyze and identify the factors associated with the pathogenicity and virulence of X.fastidiosa strain 9a5c and Fb7. To visualize the presence of LesA in the total and secreted protein samples of strains 9a5c and Fb7, esterase and lipase tests were performed. Lipase activity was analyzed by culturing the bacteria in culture medium supplemented with 1% tributyrin. This test demonstrated activity under the short chain triacylglycerols present in the medium and can be visualized by the increased halo formation in the Fb7 strain. The esterase activity, determined by the substrate 4-methylumbelliferylbutyrate (4-MUB), showed significant and clearly expressed values. Hence, the Fb7 strain showed considerably higher activity when compared to 9a5c. In search for virulence factors, a liquid chromatography coupled to mass spectrometry (LC-MS / MS) of the total proteins of the X.fastidiosa cultures of both strains was performed. In this analysis, it was identified 119 differentially expressed proteins. Among them, some proteins with functions associated with bacterial virulence were overexpressed in Fb7 strain, compared to 9a5c strain. The RT-qPCR analysis allowed to validate the expression of some of these proteins. The results showed that LesA and other proteins with higher levels of expression in Fb7 play an important pathogenicity role in this strain. Thus, they are factors that may explain the greater virulence of Fb7 strain when compared to strain 9a5c. Dissertação (Mestrado)

Made available in DSpace on 2019-10-04T12:41:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-09-04 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Brazilian Funding Agency: Foundation for Support to Teaching, Research and Assistance - FAEPA of the HCFMRP-USP The zinc finger transcription factor PAC-3/RIM101/PacC has a defined role in the secretion of enzymes and proteins in response to ambient pH, and also contributes to the virulence of species. Herein we evaluated the role of PAC-3 in the regulation of Neurospora crassa genes, in a model that examined the plant-fungi interactions. N. crassa is a model fungal species capable of exhibiting dynamic responses to its environment by employing endophytic or phytopathogenic behavior according to a given circumstance. Since plant growth and productivity are highly affected by pH and phosphorus (P) acquisition, we sought to verify the impact that induction of a Delta pac-3 mutation would have under limited and sufficient Pi availability, while ensuring that the targeted physiological adjustments mimicked ambient pH and nutritional conditions required for efficient fungal growth and development. Our results suggest direct regulatory functions for PAC-3 in cell wall biosynthesis, homeostasis, oxidation-reduction processes, hydrolase activity, transmembrane transport, and modulation of genes associated with fungal virulence. Pi-dependent modulation was observed mainly in genes encoding for transporter proteins or related to cell wall development, thereby advancing the current understanding regarding colonization and adaptation processes in response to challenging environments. We have also provided comprehensive evidence that suggests a role for PAC-3 as a global regulator in plant pathogenic fungi, thus presenting results that have the potential to be applied to various types of microbes, with diverse survival mechanisms. Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Genet, Ribeirao Preto, Brazil Morgana Potrich Coll, Dept Biofunct, Mineiros, Brazil Sao Paulo State Univ, Inst Chem, Dept Biochem & Technol Chem, UNESP, Araraquara, Brazil Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Biochem & Immunol, Ribeirao Preto, Brazil Sao Paulo State Univ, Inst Chem, Dept Biochem & Technol Chem, UNESP, Araraquara, Brazil FAPESP: 2014/03847-7 FAPESP: 2018/11319-1 FAPESP: 2016/04274-6 CNPq: 305252/2013-5 CNPq: 304222/2013-5 CAPES: 001

Plant diseases caused by fungal pathogens are responsible for major crop losses worldwide, with a significant socio-economic impact on the life of millions of people who depend on agriculture-exclusive economy. This is the case of the Witches' Broom Disease (WBD) affecting cacao plant and fruit in South and Central America. The severity and extent of this disease is prospected to impact the growing global chocolate market in a few decades. WBD is caused by the basidiomycete fungus Moniliophthora perniciosa. The methods used to contain the fungus mainly rely on chemical fungicides, such as copper-based compounds or azoles. Not only are these highly ineffective, but also their utilization is increasingly restricted by the cacao industry, in part because it promotes fungal resistance, in part related to consumers' health concerns and environmental awareness. Therefore, the disease is being currently tentatively controlled through phytosanitary pruning, although the full removal of infected plant material is impossible and the fungus maintains persistent inoculum in the soil, or using an endophytic fungal parasite of Moniliophthora perniciosa which production is not sustainable. The growth of Moniliophthora perniciosa was reported as being antagonized in vitro by some yeasts, which suggests that they could be used as biological control agents, suppressing the fungus multiplication and containing its spread. Concurrently, some yeast-based products are used in the protection of fruits from postharvest fungal spoilage, and the extension of diverse food products shelf-life. These successful applications suggest that yeasts can be regarded a serious alternative also in the pre-harvest management of WBD and other fungal plant diseases. Yeasts' GRAS (Generally Recognized as Safe) nature adds to their appropriateness for field application, not raising major ecological concerns as do the present more aggressive approaches. Importantly, mitigating WBD, in a sustainable manner, would predictably have a high socioeconomic impact, contributing to diminish poverty in the cacao-producing rural communities severely affected by the disease. This review discusses the importance/advantages and the challenges that such a strategy would have for WBD containment, and presents the available information on the molecular and cellular mechanisms underlying fungi antagonism by yeasts., This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI), and the project EcoAgriFood (NORTE-01-0145-FEDER-000009), supported by the Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF). PF is a Ph.D. student of the Doctoral Programme in Applied and Environmental Microbiology (DP_AEM) and FCT grantee PD/BD/113810/2015.

Pathogenic bacteria use Quorum sensing (QS) to regulate the expression of virulence factors involved in plant tissue infection. Some of these factors are the production of biofilm, hydrolytic enzymes, toxins, and plasmids; therefore, the interruption of this system could be a useful tool to control plant tissue infections. This review analyzes the potential treatments to interrupt QS and control the infection of plant tissues.

El cultivo comercial de hortensias para flor de exportación ocupa un renglón importante en el sector económico del oriente antioqueño, por ser fuente de empleo y de desarrollo en la zona. La hortensia (Hydrangea macrophylla) es afectada por numerosos organismos fitopatógenos, entre ellos, nematodos del género Aphelenchoides, los que ocasionan, en el follaje, lesiones necróticas angulares, malformación de flor, enanismo y un daño indirecto en la tasa fotosintética, demeritando los parámetros de calidad para exportación. El objetivo de este estudio fue identificar, molecularmente, las especies del nematodo Aphelenchoides asociadas al cultivo de hortensias de color, en los municipios de Medellín (Santa Elena), La Ceja y Rionegro, siendo este el primer reporte para Colombia, de las especies de este género. Para la ejecución del estudio, se realizaron 10 muestreos en cultivos comerciales, distribuidos entre los tres municipios mencionados. Los nematodos extraídos, se sometieron a pruebas basadas en el análisis de ADN, haciendo uso del marcador ribosomal 18S. Los análisis filogenéticos practicados mostraron la presencia de la especie Aphelenchoides ritzemabosi en cultivos de hortensias, del corregimiento de Santa Elena y, de A. fragarie, en los municipios de La Ceja y Rionegro. Hydrangeamacrophylla commercial crops have an import-ant economic significance for the eastern region of the de-partment of Antioquia, Colombia, due to its impact on em-ployment and local development. This flowering plant can be attacked by several phytopathogens among them nem-atodes of the genus Aphelenchoides; which can produce necrotic lesions on the plant leaves as well decreased photo-synthetic rate, dwarfism, and flower malformation. Addition-ally, this pathogen is considered a regulated pest and limits the international commercialization of the flower. The aim of this study was to molecularly identify the Aphelenchoidesspecies associated with infected H.macrophylla plants in crops the municipalities of Medellín, La Ceja, and Rionegro. This is the first report of the species of this phytopathogen for Colombia. The sampling activities were performed in ten commercial crops locate at the three municipalities. The isolated nematodes were subject to DNA-based tests were the 18S rDNA gene was amplified, sequenced and analyzed using phylogenetic methods. The obtained results showed the infection of Aphelenchoides ritzemabosi within the Santa Elena (Medellin) area in crops and A. fragarie within the mu-nicipalities of La Ceja, and Rionegro. Incluye referencias bibliográficas

Type IV pili (Tfp) are widely distributed adhesins of bacterial surfaces. In plant pathogenic bacteria, Tfp are involved in host colonization and pathogenesis. Xanthomonas citri subsp. citri (Xcc) is the phytopathogen responsible for citrus canker disease. In this work, three Tfp structural genes, fimA, fimA1, and pilA from Xcc were studied. A pilA mutant strain from Xcc (XccΔpilA) was constructed and differences in physiological features, such as motilities, adhesion, and biofilm formation, were observed. A structural study of the purified Tfp fractions from Xcc wild-type and Xcc∆pilA showed that pilins are glycosylated in both strains and that FimA and FimA1 are the main structural components of the pili. Furthermore, smaller lesion symptoms and reduced bacterial growth were produced by Xcc∆pilA in orange plants compared to the wild-type strain. These results indicate that the minor pilin-like gene, pilA, is involved in Tfp performance during the infection process. Fil: Petrocelli, Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Arana, Maite Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Cabrini, Marcela N.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Casabuono, Adriana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Moyano, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Beltramino, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Moreira, Leandro M.. Universidade Federal de Ouro Preto; Brasil Fil: Couto, Alicia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Orellano, Elena Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Bacillus thuringiensis subsp. tenebrionis DSM‐2803 has been studied extensively and spore/crystal mixtures of this strain are used widely in commercial products to control coleopteran pests. The endochitinase chiA Btt gene of B. thuringiensis subsp. tenebrionis DSM‐2803 was cloned and expressed in Escherichia coli. The recombinant 6x‐histidine tagged protein (rChiA Btt, ~74 kDa), was purified by a HiTrap Ni affinity column. The Km of rChiA Btt was 0.847 μmol L−1 and its optimal activity occurred at pH 7 and ~40°C. Most divalent cations reduced endochitinase activity but only Hg+2 abolished activity of the enzyme. We report for the first time the characterization of a chitinase synthesized by B. thuringiensis subsp. tenebrionis DSM‐2803, and show that the purified rChiA74 Btt reduced the radial growth and increased the hyphal density of Colletotrichium gloeosporioides, the etiological agent of “anthracnose” in plants.

RESUMOO feijão-fava (Phaseolus lunatus L.) tem se mostrado uma importante fonte alternativa de alimentação e renda para as populações mais carentes na região Nordeste do Brasil. Entretanto, vários fatores, dentre eles a ocorrência de doenças têm contribuído para o baixo rendimento da cultura. Assim, no presente trabalho objetivou-se avaliar a reação de doze acessos de feijão-fava, de hábito de crescimento determinado, ao agente causal da antracnose, Colletotrichum truncatum, em condições de folha destacada, inoculada com o fungo e campo, sem inoculação. A severidade da doença foi estimada através de escala de notas e os acessos foram agrupados em cinco classes de acordo com a resistência. Os acessos apresentaram resistência variável à antracnose, de acordo com as condições de cultivo, sendo que em folhas destacadas foram mais suscetíveis ao fungo do que no campo. Observou-se no campo, para a maioria dos acessos, redução na porcentagem de área foliar doente em comparação com as respostas apresentadas em folha destacada. Os acessos UFPI 641, UFPI 644 e UFPI 645 comportaram-se como moderadamente resistentes em folha destacada, aos sete dias após a inoculação. Estes genótipos são, portanto, promissores para serem utilizados em programas de melhoramento genético do feijão-fava. ABSTRACTThe lima bean (Phaseolus lunatus L.) has become an important alternative source of food and earnings to low-income populations in the Northeast Region of Brazil. However, several factors have contributed to the low yield of this crop, such as occurrence of diseases. Thus, the present study aimed to assess the reaction of twelve lima bean accessions, of determined growth habit, to the causal agent of anthracnose, Colletotrichum truncatum, under conditions of detached leaves inoculated with the fungus and in the field without inoculation. The severity of the disease was estimated by means of a score scale and the accessions were grouped into five classes according to resistance. The accessions had variable resistance to anthracnose according to the growth conditions. Detached leaves were more susceptible to the fungus than those in the field. For most accessions in the field, there was a reduction in the percentage of diseased leaf area, compared to the responses shown by detached leaves. The accessions UFPI 641, UFPI 644 and UFPI 645 behaved as moderately resistant for detached leaves at seven days after inoculation. These genotypes are, therefore, promising for breeding programs of lima beans.