Dataset Information

Association of Lasiodiplodia theobromae with die-back and decline of nutmeg as revealed through phenotypic, pathogenicity and phylogenetic analyses.

ABSTRACT: Lasiodiplodia theobromae is a cosmopolitan pathogen geographically widespread in tropics and subtropics inciting economically important diseases on diverse plant genera. In the present study, Lasiodiplodia theobromae associated with nutmeg exhibiting die-back and declining symptoms was identified and characterized by adopting a polyphasic approach. The disease was characterized with the symptoms including general decline, water-soaking patches on branches and tree trunk, die-back of branches, necrotic lesions beneath water-soaked lesions and necrosis of vascular tissues. The isolates representing diverse nutmeg growing tracts were initially identified as Lasiodiplodia species based on macro- and micro-morphological characteristics. Subsequent analyses of internal transcribed spacer (ITS), partial elongation factor 1-alpha (EF1-α) and β-tubulin (β-tub) genes identified the pathogen as Lasiodiplodia theobromae. Pathogenicity studies were proved on nutmeg twigs and branches (in vitro) as well as on saplings (in vivo). The present investigation enunciated the association of Lasiodiplodia theobromae with die-back and decline of nutmeg employing a polyphasic approach which warrants further investigations on its spatio-temporal distribution, pathogen diversity, weather-host-pathogen interaction and formulating prospective disease management strategies.

SUBMITTER: Biju CN

PROVIDER: S-EPMC8410934 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Association of Lasiodiplodia theobromae with die-back and decline of nutmeg as revealed through phenotypic, pathogenicity and phylogenetic analyses.

Biju C N CN Jeevalatha A A Peeran M F MF Bhai R Suseela RS Basima Fadla F Nissar V A Muhammed VAM Srinivasan V V Thomas Lijo L

3 Biotech 20210901 9

Lasiodiplodia theobromae is a cosmopolitan pathogen geographically widespread in tropics and subtropics inciting economically important diseases on diverse plant genera. In the present study, Lasiodiplodia theobromae associated with nutmeg exhibiting die-back and declining symptoms was identified and characterized by adopting a polyphasic approach. The disease was characterized with the symptoms including general decline, water-soaking patches on branches and tree trunk, die-back o ...[more]

PMID: 34603922

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Project description:BackgroundLasiodiplodia theobromae is a fungus of the Botryosphaeriaceae that causes grapevine vascular disease, especially in regions with hot climates. Fungi in this group often remain latent within their host and become virulent under abiotic stress. Transcriptional regulation analysis of L. theobromae exposed to heat stress (HS) was first carried out in vitro in the presence of grapevine wood (GW) to identify potential pathogenicity genes that were later evaluated for in planta expression.ResultsA total of 19,860 de novo assembled transcripts were obtained, forty-nine per cent of which showed homology to the Botryosphaeriaceae fungi, Neofusicoccum parvum or Macrophomina phaseolina. Three hundred ninety-nine have homology with genes involved in pathogenic processes and several belonged to expanded gene families in others fungal grapevine vascular pathogens. Gene expression analysis showed changes in fungal metabolism of phenolic compounds; where genes encoding for enzymes, with the ability to degrade salicylic acid (SA) and plant phenylpropanoid precursors, were up-regulated during in vitro HS response, in the presence of GW. These results suggest that the fungal L-tyrosine catabolism pathway could help the fungus to remove phenylpropanoid precursors thereby evading the host defense response. The in planta up-regulation of salicylate hydroxylase, intradiol ring cleavage dioxygenase and fumarylacetoacetase encoding genes, further supported this hypothesis. Those genes were even more up-regulated in HS-stressed plants, suggesting that fungus takes advantage of the increased phenylpropanoid precursors produced under stress. Pectate lyase was up-regulated while a putative amylase was down-regulated in planta, this could be associated with an intercellular growth strategy during the first stages of colonization.ConclusionsL. theobromae transcriptome was established and validated. Its usefulness was demonstrated through the identification of genes expressed during the infection process. Our results support the hypothesis that heat stress facilitates fungal colonization, because of the fungus ability to use the phenylpropanoid precursors and SA, both compounds known to control host defense.

Project description:Purpose: Lasiodiplodia theobromae is a pathogenic fungus causing Botryosphaeria dieback in grapevine. Heat and drought stress was suggested to promote the disease incidence in recent years, therefore we decided to evaluate the effect of heat on in vitro experiment, adding grapevine wood to simulate the environment in the trunk during in field infection. Methods: Fungal mRNA was extracted from mycellium collected after 49 hours of growth without stress in the presence of GW (GW_noHS) or in its absence (VS_noHS). Some mRNA were extracted form mycellium collected 10 min after stress (1 hour at 42 C) both in the presence of GW (GW_HS) or in its absence (VS_HS). The cDNA and sequenciation was done according with Illumina HiSeq 2500 recomendations by triplicate. Paired end reads (100bp) were used for de novo assembly through Trinity. Transdecoder was used to predict ORFs. The transcripts functional annotatio was done through BlastX and Blast2GO. Bowtie2 was used to map the reads for each sample to the previously assembled transcritome, a homemade Perl script was used to count the mapped reads and edgeR was employed for differential expression and statistical analysis. Results: Seventy million reads were asembled to produce 19,860 transcripts, from those 15,981 were predicted to codify for ORFs. Thirty-five % of transcripts did not have any homologue in NCBI NR database, most of those also did not were predicted to codify for ORFs. With the remainning 38 % of transcripts the functional annotation was obtained. Around 90% of reads for each sample were mapped to the de novo assembled transcriptome. Around 2339 genes were differentially expressed when comparing GW_HS vs GW_noHS or vs VS_noHS and GW_noHS vs VS_noHS and VS_HS vs VS_noHS. Hierarchical clustering with functional enrichment were used to predict metabolic pathways with differential regulation. In silico prediction were then verified with RT-qPCR showing linear correlation (r2=0.87) in in vitro experiment. Then trough in planta fungal gene expression helps to verify that phenolic metabolism is important for colonization in grapevine. Conclusion: A model for fungal response to HS in the presence of GW was proposed. This correspond to the first work to identify nucleotide sequences, measure gene expression and propose pathogenicity factors important for disease establishment in grapevine.

Project description:Environmental alterations modulate host-microorganism interactions. Little is known about how climate changes can trigger pathogenic features on symbiont or mutualistic microorganisms. Current climate models predict increased environmental temperatures. The exposing of phytopathogens to these changing conditions can have particularly relevant consequences for economically important species and for humans. The impact on pathogen/host interaction and the shift on their biogeographical range can induce different levels of virulence in new hosts, allowing massive losses in agricultural and health fields. Lasiodiplodia theobromae is a phytopathogenic fungus responsible for a number of diseases in various plants. It has also been described as an opportunist pathogen in humans, causing infections with different levels of severity. L. theobromae has a high capacity of adaptation to different environments, such as woody plants, moist argillaceous soils, or even humans, being able to grow and infect hosts in a wide range of temperatures (9-39°C). Nonetheless, the effect of an increase of temperature, as predicted in climate change models, on L. theobromae is unknown. Here we explore the effect of temperature on two strains of L. theobromae - an environmental strain, CAA019, and a clinical strain, CBS339.90. We show that both strains are cytotoxic to mammalian cells but while the environmental strain is cytotoxic mainly at 25°C, the clinical strain is cytotoxic mainly at 30 and 37°C. Extracellular gelatinolytic, xylanolytic, amylolytic, and cellulolytic activities at 25 and 37°C were characterized by zymography and the secretome of both strains grown at 25, 30, and 37°C were characterized by electrophoresis and by Orbitrap LC-MS/MS. More than 75% of the proteins were identified, mostly enzymes (glycosyl hydrolases and proteases). The strains showed different protein profiles, which were affected by growth temperature. Also, strain specific proteins were identified, such as a putative f5/8 type c domain protein - known for being involved in pathogenesis - by strain CAA019 and a putative tripeptidyl-peptidase 1 protein, by strain CBS339.90. We showed that temperature modulates the secretome of L. theobromae. This modulation may be associated with host-specificity requirements. We show that the study of abiotic factors, such as temperature, is crucial to understand host/pathogen interactions and its impact on disease.

Dataset Information

Association of Lasiodiplodia theobromae with die-back and decline of nutmeg as revealed through phenotypic, pathogenicity and phylogenetic analyses.

Publications

Association of <i>Lasiodiplodia theobromae</i> with die-back and decline of nutmeg as revealed through phenotypic, pathogenicity and phylogenetic analyses.

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