scholarly journals Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene, AvrLmS-Lep2

2020 ◽  
Author(s):  
Ting Xiang Neik ◽  
Kaveh Ghanbarnia ◽  
Bénédicte Ollivier ◽  
Armin Scheben ◽  
Anita Severn-Ellis ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Leptosphaeria Maculans ◽  
Lesion Size ◽  
Avirulence Gene ◽  
Avirulence Genes ◽  
Blackleg Disease ◽  
Fungal Plant Pathogens ◽  
Potential Efficiency ◽  
Secretory Signal Peptide

SummaryLeptosphaeria maculans, the causal agent of blackleg disease, interacts with Brassica napus (oilseed rape, canola) in a gene-for-gene manner. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in the cultivar Surpass 400. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (Bulked-Segregant-Sequencing) to rapidly identify one candidate avirulence gene present in the effector repertoire of L. maculans. AvrLep2 was cloned using a bi-parental cross of avirulent and virulent L. maculans isolates and a classical map-based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400 and Topas-LepR2. The gene renamed AvrLmS-Lep2 encodes for a small cysteine-rich protein of unknown function with an N-terminal secretory signal peptide, which are common features of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS-Lep2 / LepR2 interaction phenotype was found to vary from a typical hypersensitive response to intermediate resistance sometimes at the edge of, or evolving toward, susceptibility depending on the inoculation conditions. AvrLmS-Lep2 was nevertheless sufficient to significantly reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.

scholarly journals Evaluation of in-vitro methods to select effective streptomycetes against toxigenic fusaria

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6905 ◽  
Author(s):  
Elena Maria Colombo ◽  
Cristina Pizzatti ◽  
Andrea Kunova ◽  
Claudio Gardana ◽  
Marco Saracchi ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Culture Media ◽  
Screening Methods ◽  
Dual Culture ◽  
In Planta ◽  
In Vitro Method ◽  
In Vitro Methods ◽  
Strain Diversity ◽  
Fungal Plant Pathogens

Biocontrol microorganisms are emerging as an effective alternative to pesticides. Ideally, biocontrol agents (BCAs) for the control of fungal plant pathogens should be selected by an in vitro method that is high-throughput and is predictive of in planta efficacy, possibly considering environmental factors, and the natural diversity of the pathogen. The purpose of our study was (1) to assess the effects ofFusariumstrain diversity (N= 5) and culture media (N= 6) on the identification of biological control activity ofStreptomycesstrains (N= 20) againstFusariumpathogens of wheat in vitro and (2) to verify the ability of our in vitro screening methods to simulate the activity in planta. Our results indicate that culture media,Fusariumstrain diversity, and their interactions affect the results of an in vitro selection by dual culture assay. The results obtained on the wheat-based culture media resulted in the highest correlation score (r= 0.5) with the in planta root rot (RR) inhibition, suggesting that this in vitro method was the best predictor of in planta performance of streptomycetes against Fusarium RR of wheat assessed as extension of the necrosis on the root. Contrarily, none of the in vitro plate assays using the media tested could appropriately predict the activity of the streptomycetes against Fusarium foot rot symptoms estimated as the necrosis at the crown level. Considering overall data of correlation, the activity in planta cannot be effectively predicted by dual culture plate studies, therefore improved in vitro methods are needed to better mimic the activity of biocontrol strains in natural conditions. This work contributes to setting up laboratory standards for preliminary screening assays ofStreptomycesBCAs against fungal pathogens.

scholarly journals Plant Defensin Peptides have Antifungal and Antibacterial Activity Against Human and Plant Pathogens

2019 ◽  
Vol 109 (3) ◽  
pp. 402-408 ◽  
Author(s):  
Andrew E. Sathoff ◽  
Siva Velivelli ◽  
Dilip M. Shah ◽  
Deborah A. Samac
Keyword(s):  
Antibacterial Activity ◽  
Plant Pathogens ◽  
Crown Rot ◽  
Plant Defensin ◽  
Core Motif ◽  
Potential Control ◽  
Fungal Plant Pathogens ◽  
Plant Defensins ◽  
Animal Pathogens

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

scholarly journals Analysis of Molecular Markers Genetically Linked to the Leptosphaeria maculans Avirulence Gene AvrLm1 in Field Populations Indicates a Highly Conserved Event Leading to Virulence on Rlm1 Genotypes

2002 ◽  
Vol 15 (7) ◽  
pp. 672-682 ◽  
Author(s):  
Agnès Attard ◽  
Lilian Gout ◽  
Mathieu Gourgues ◽  
Marie-Line Kühn ◽  
Jacques Schmit ◽  
...  
Keyword(s):  
Genetic Map ◽  
Large Scale ◽  
Polymerase Chain Reaction Amplification ◽  
Leptosphaeria Maculans ◽  
Avirulence Gene ◽  
Bac Contig ◽  
Genome Region ◽  
Repulsion Phase ◽  
Field Isolates

Map-based cloning of the avirulence gene AvrLm1 of Leptosphaeria maculans was initiated utilizing a genetic map of the fungus and a BAC library constructed from an AvrLm1 isolate. Seven polymorphic DNA markers closely linked to AvrLm1 were identified. Of these, two were shown to border the locus on its 5′ end and were present, with size polymorphism, in both the virulent and the avirulent isolates. In contrast, three markers, J19-1.1, J53-1.3 (in coupling phase with avirulence), and Vir1 (in repulsion phase with avirulence), cosegregated with AvrLm1 in 312 progeny from five in vitro crosses. J19-1.1 and J53-1.3 were never amplified in the virulent parents or progeny, whereas Vir1 was never amplified in the avirulent parents or progeny. J19-1.1 and J53-1.3 were shown to be separated by 40 kb within a 184-kb BAC contig. In addition, the 1.6-cM genetic distance between J53-1.3 and the nearest recombinant marker corresponded to a 121-kb physical distance. When analyzing a European Union-wide collection of 192 isolates, J53-1.3, J19-1.1, and Vir1 were found to be closely associated with the AvrLm1 locus. The results of polymerase chain reaction amplification with primers for the three markers were in accordance with the interaction phenotype for 92.2% (J53-1.3), 90.6% (J19-1.1), and 88.0% (Vir1) of the isolates. In addition, genome organization of the AvrLm1 region was highly conserved in field isolates, because 89.1% of the avirulent isolates and 79.0% of the virulent isolates showed the same association of markers as that of the parents of in vitro crosses. The large-scale analysis of field isolates with markers originating from the genetic map therefore confirms (i) the physical proximity between the markers and the target locus and (ii) that AvrLm1 is located in (or close to) a recombination-deficient genome region. As a consequence, map-based markers provided us with high-quality markers for an overview of the occurrence of race “AvrLm1” at the field scale. These data were used to propose hypotheses on evolution towards virulence in field isolates.

Antifungal activity of extracts from endophytic fungi associated with Smallanthus maintained in vitro as autotrophic cultures and as pot plants in the greenhouse

2012 ◽  
Vol 58 (10) ◽  
pp. 1202-1211 ◽  
Author(s):  
Luiz H. Rosa ◽  
Nurhayat Tabanca ◽  
Natascha Techen ◽  
Zhiqiang Pan ◽  
David E. Wedge ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Endophytic Fungi ◽  
Plant Pathogens ◽  
Cultivation Conditions ◽  
Smallanthus Sonchifolius ◽  
Fungal Plant Pathogens ◽  
In The Wild ◽  
Fungal Assemblages ◽  
Pot Plants

The endophytic fungal assemblages associated with Smallanthus sonchifolius (Poepp.) H. Rob. and Smallanthus uvedalius (L.) Mack. ex Small growing in vitro autotrophic cultures and in the greenhouse were identified and evaluated for their ability to produce bioactive compounds. A total of 25 isolates were recovered that were genetically closely related to species of the genera Bionectria , Cladosporium , Colletotrichum , Fusarium , Gibberella , Hypocrea , Lecythophora , Nigrospora , Plectosphaerella , and Trichoderma . The endophytic assemblages of S. sonchifolius presented a greater diversity than the group isolated from S. uvedalius and demonstrated the presence of dominant generalist fungi. Extracts of all fungi were screened against the fungal plant pathogens. Ten extracts (41.6%) displayed antifungal activities; some of them had a broad antifungal activity. The phylotypes Lecythophora sp. 1, Lecythophora sp. 2, and Fusarium oxysporum were isolated from in vitro autotrophic cultures and displayed antifungal activity. The presence of bioactive endophytic fungi within S. sonchifolius and S. uvedalius suggests an ecological advantage against pathogenic attacks. This study revealed reduced numbers of endophytes in association with both Smallanthus species in controlled cultivation conditions compared with the endophytic communities of hosts collected in the wild environments. Even as reduced endophytic communities, these fungi continue to provide chemical protection for the host.

Douglas-fir root-associated microorganisms with inhibitory activity towards fungal plant pathogens and human bacterial pathogens

1996 ◽  
Vol 42 (7) ◽  
pp. 690-700 ◽  
Author(s):  
Paige E. Axelrood ◽  
Alison M. Clarke ◽  
Reed Radley ◽  
S. Janet V. Zemcov
Keyword(s):  
Antifungal Activity ◽  
Inhibitory Activity ◽  
Plant Pathogens ◽  
Bacterial Pathogens ◽  
Douglas Fir ◽  
Microbial Culture ◽  
Bacterial Strains ◽  
Fungal Plant Pathogens ◽  
Forest Sites

A microbial culture collection composed of 1820 bacterial strains, including 298 actinomycete strains, was established from the roots of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings harvested from conifer nurseries and forest sites. Two hundred and thirty-four strains inhibited the growth of Fusarium, Cylindrocarpon, and (or) Pythium spp. in in vitro assays. A significantly greater proportion of bacterial strains from actinomycete genera exhibited antifungal properties compared with bacterial strains from nonactinomycete genera. Eighty-nine percent of identified inhibitory strains were Streptomyces, Streptoverticillium, Bacillus, Pseudomonas, or Burkholderia species. The actinomycete species were isolated almost exclusively from forest seedlings. Recovery of inhibitory strains representing 29 microbial species was enhanced using a variety of methods to isolate microorganisms from the roots of seedlings from nursery and forest sites. Bacterial strains (including actinomycete strains) with antifungal activity were tested for in vitro growth inhibition of six clinical human bacterial pathogens (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa). Forty-eight percent of the tested strains inhibited one or more human pathogens. Inhibitory activity towards fungal and bacterial pathogens was strain specific, not species specific, and many inhibitory strains exhibited broad-spectrum activity. Strains with antifungal activity against several conifer root pathogens were also more likely to inhibit multiple species of clinical bacterial pathogens.Key words: in vitro, antimicrobial, conifer rhizosphere.

scholarly journals Sensitivity of Bacterial and Fungal Plant Pathogens to the Lytic Peptides, MSI-99, Magainin II, and Cecropin B

2002 ◽  
Vol 15 (7) ◽  
pp. 701-708 ◽  
Author(s):  
Ali R. Alan ◽  
Elizabeth D. Earle
Keyword(s):  
Phytophthora Infestans ◽  
Plant Pathogens ◽  
Fungal Spores ◽  
Alternaria Solani ◽  
Leaf Disk ◽  
Cecropin B ◽  
Fungal Plant Pathogens ◽  
Magainin Ii ◽  
Lytic Peptides

In vitro and leaf disk assays of bacterial and fungal plant pathogens were conducted using three cationic lytic peptides, MSI-99, magainin II (MII), and cecropin B (CB). Growth of bacterial organisms was retarded or completely inhibited by low concentrations of these lytic peptides. The peptides also significantly reduced germination of fungal spores and growth of mycelia; however, higher concentrations of peptides were needed to inhibit fungal growth compared with those needed to inhibit bacteria. The relative efficacy of the peptides depended on the microorganism tested, but CB was the most inhibitory to the majority of the bacteria and fungi assayed. MSI-99, a synthetic derivative of MII with increased positive charge, showed equal or two- to fivefold higher antibacterial activity compared to MII in the in vitro assays. MSI-99 was also superior to MII against the oomycete, Phytophthora infestans but was slightly inferior to MII in assays with the true fungi, Penicillium digitatum and Alternaria solani. In the leaf disk assays, pretreating spores of Alternaria solani and Phytophthora infestans with the peptides at concentrations as low as 10 μg per ml led to significant reductions in the size of early blight lesions and prevented development of any late blight lesions on tomato leaf disks. Our results from in vitro and leaf disk assays suggest that MSI-99 can be used as a transgene to generate tomato lines with enhanced resistance to bacterial and fungal diseases of this crop.

Classification of airborne plant pathogens based on sporulation and infection characteristics

1995 ◽  
Vol 73 (8) ◽  
pp. 1186-1195 ◽  
Author(s):  
Ivan Sache ◽  
Claude de Vallavieille-Pope
Keyword(s):  
Plant Pathogens ◽  
Lesion Size ◽  
The Other ◽  
Infectious Period ◽  
Downy Mildews ◽  
Fungal Plant Pathogens ◽  
Comparative Epidemiology ◽  
Lesion Growth ◽  
Sporulation Capacity ◽  
Epidemiological Characteristics

The infection cycles of 26 airborne fungal plant pathogens were compared using six monocyclic variables: latent period, infectious period, sporulation capacity, relative date of sporulation peak, lesion size, and infection efficiency. All variables were measured at the seedling stage in conditions highly conducive to disease development. Multivariate analyses of literature and experimental data were used to describe epidemic strategies based on compensation, addition, and multiplication effects between the monocyclic variables. A typology of fungi according to these effects is proposed, the main divisions of which follow the pattern of lesion growth (semisystemic, local, or negligible). Further subdivisions were obtained based on the other sporulation and infection variables. With a few exceptions, pathogens belonging to the same taxonomic (powdery mildews, downy mildews, rusts) and ecological (biotrophs, necrotrophs) group were grouped together in the proposed typology. Among rusts and necrotrophs, the fungi adapted to temperate and warm climates were also distinguished. The wheat stripe rust was not grouped with the other rusts because of unique epidemiological characteristics, especially semisystemic growth of lesions. Key words: biotrophic fungus, comparative epidemiology, monocyclic process, necrotrophic fungus.

scholarly journals Transfection of Sclerotinia sclerotiorum withIn VitroTranscripts of a Naturally Occurring Interspecific Recombinant of Sclerotinia sclerotiorum Hypovirus 2 Significantly Reduces Virulence of the Fungus

2015 ◽  
Vol 89 (9) ◽  
pp. 5060-5071 ◽  
Author(s):  
Shin-Yi Lee Marzano ◽  
Houston A. Hobbs ◽  
Berlin D. Nelson ◽  
Glen L. Hartman ◽  
Darin M. Eastburn ◽  
...  
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Plant Pathogens ◽  
High Throughput Sequencing ◽  
Infectious Clone ◽  
White Mold ◽  
Content Type ◽  
Fungal Plant Pathogens ◽  
Rapid Amplification ◽  
Valsa Ceratosperma

ABSTRACTA recombinant strain ofSclerotinia sclerotiorumhypovirus 2 (SsHV2) was identified from a North AmericanSclerotinia sclerotiorumisolate (328) from lettuce (Lactuca sativaL.) by high-throughput sequencing of total RNA. The 5′- and 3′-terminal regions of the genome were determined by rapid amplification of cDNA ends. The assembled nucleotide sequence was up to 92% identical to two recently reported SsHV2 strains but contained a deletion near its 5′ terminus of more than 1.2 kb relative to the other SsHV2 strains and an insertion of 524 nucleotides (nt) that was distantly related toValsa ceratospermahypovirus 1. This suggests that the new isolate is a heterologous recombinant of SsHV2 with a yet-uncharacterized hypovirus. We named the new strainSclerotinia sclerotiorumhypovirus 2 Lactuca (SsHV2L) and deposited the sequence in GenBank with accession numberKF898354.Sclerotinia sclerotiorumisolate 328 was coinfected with a strain ofSclerotinia sclerotiorumendornavirus 1 and was debilitated compared to cultures of the same isolate that had been cured of virus infection by cycloheximide treatment and hyphal tipping. To determine whether SsHV2L alone could induce hypovirulence inS. sclerotiorum, a full-length cDNA of the 14,538-nt viral genome was cloned. Transcripts corresponding to the viral RNA were synthesizedin vitroand transfected into a virus-free isolate ofS. sclerotiorum, DK3. Isolate DK3 transfected with SsHV2L was hypovirulent on soybean and lettuce and exhibited delayed maturation of sclerotia relative to virus-free DK3, completing Koch's postulates for the association of hypovirulence with SsHV2L.IMPORTANCEA cosmopolitan fungus,Sclerotinia sclerotioruminfects more than 400 plant species and causes a plant disease known as white mold that produces significant yield losses in major crops annually. Mycoviruses have been used successfully to reduce losses caused by fungal plant pathogens, but definitive relationships between hypovirus infections and hypovirulence inS. sclerotiorumwere lacking. By establishing a cause-and-effect relationship betweenSclerotinia sclerotiorumhypovirus Lactuca (SsHV2L) infection and the reduction in host virulence, we showed direct evidence that hypoviruses have the potential to reduce the severity of white mold disease. In addition to intraspecific recombination, this study showed that recent interspecific recombination is an important factor shaping viral genomes. The construction of an infectious clone of SsHV2L allows future exploration of the interactions between SsHV2L andS. sclerotiorum, a widespread fungal pathogen of plants.

scholarly journals Diversity of biocontrol agents, isolated from several sources, inhibitory to several fungal plant pathogens

2020 ◽  
Vol 22 (1) ◽  
Author(s):  
Yan Ramona ◽  
IDA BAGUS GEDE DARMAYASA ◽  
ANAK AGUNG NGURAH NARA KUSUMA ◽  
Martin Line
Keyword(s):  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Antagonistic Activity ◽  
Significant Inhibition ◽  
Biocontrol Agents ◽  
Dual Culture ◽  
Rdna Sequences ◽  
Fungal Plant Pathogens ◽  
Mature Compost

Abstract. Ramona Y, Darmayasa IBG, Kusuma AANN, Line MA. 2021. Diversity of biocontrol agents, isolated from several sources, inhibitory to several fungal plant pathogens. Biodiversitas 22: 298-303. This study investigated the inhibitory potential of diversity of antagonist bacteria residing in the rhizosphere zone and mature compost to counter fungal plant pathogens. Soils collected from rhizosphere of lettuce farms in Bali-Indonesia and Tasmania-Australia, mature compost, commercial biocontrol (Dipel®), and laboratory contaminants with significant inhibition against tested fungal pathogens were used as sources of antagonist bacteria. These antagonists were isolated by applying dilution and spread method on trypticase soya agar (TSA) or potato dextrose agar (PDA), and their ability to inhibit Sclerotinia minor, Sclerotinia sclerotiorum, Fusarium spp., and Rhizoctonia solani was assessed in dual culture assays. The results showed that 67 out of more than 100 isolates had antagonistic activity in vitro against at least one of tested fungal pathogens. In the preliminary identification, Bacillus spp. or Pseudomonas spp. were found to be pre-dominant isolates. Following screening studies in a non-replicated glasshouse experiment against S. minor and S. sclerotiorum, 8 of the most promising isolates were further identified using molecular methods based on their 16s rDNA sequences aligned with those deposited at the GeneBank. These 8 isolates were identified as Pseudomonas corrugata, Bacillus megaterium, Bacillus polymyxa, Bacillus mojavensis, Bacillus pumilus, Bacillus thuringiensis, Exiguobacterium acetylicum, and Chryseobacterium indologenes.

scholarly journals Constitutive expression of transcription factor SirZ blocks pathogenicity in Leptosphaeria maculans independently of sirodesmin production

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252333
Author(s):  
Andrew S. Urquhart ◽  
Candace E. Elliott ◽  
Wei Zeng ◽  
Alexander Idnurm
Keyword(s):  
Transcription Factor ◽  
Pathogenic Fungus ◽  
Leptosphaeria Maculans ◽  
Constitutive Expression ◽  
Blackleg Disease ◽  
Gene Encoding ◽  
Over Expression ◽  
Expression Construct ◽  
New Strategies

Sirodesmin, the major secondary metabolite produced by the plant pathogenic fungus Leptosphaeria maculans in vitro, has been linked to disease on Brassica species since the 1970s, and yet its role has remained ambiguous. Re-examination of gene expression data revealed that all previously described genes and two newly identified genes within the sir gene cluster in the genome are down-regulated during the crucial early establishment stages of blackleg disease on Brassica napus. To test if this is a strategy employed by the fungus to avoid damage to and then detection by the host plant during the L. maculans asymptomatic biotrophic phase, sirodesmin was produced constitutively by overexpressing the sirZ gene encoding the transcription factor that coordinates the regulation of the other genes in the sir cluster. The sirZ over-expression strains had a major reduction in pathogenicity. Mutation of the over-expression construct restored pathogenicity. However, mutation of two genes, sirP and sirG, required for specific steps in the sirodesmin biosynthesis pathway, in the sirZ over-expression background resulted in strains that were unable to synthesize sirodesmin, yet were still non-pathogenic. Elucidating the basis for this pathogenicity defect or finding ways to overexpress sirZ during disease may provide new strategies for the control of blackleg disease.

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