Transcriptional insights of citrus defense response against Diaporthe citri.

Citrus melanose, caused by Diaporthe citri, is one of the most important and widespread fungal diseases of citrus. Previous studies demonstrated that the citrus host was able to trigger the defense response to restrict the spread of D. citri. However, the molecular mechanism underlying this defense response has yet to be elucidated. Here, we used RNA-Seq to explore the gene expression pattern at the early (3 days post infection, dpi) and late (14 dpi) infection stages of citrus leaves in response to D. citri infection, and outlined the differences in transcriptional regulation associated with defense responses. The functional enrichment analysis indicated that the plant cell wall biogenesis was significantly induced at the early infection stage, while the callose deposition response was more active at the late infection stage. CYP83B1 genes of the cytochrome P450 family were extensively induced in the callus deposition-mediated defense response. Remarkably, the gene encoding pectin methylesterase showed the highest upregulation and was only found to be differentially expressed at the late infection stage. Genes involved in the synthesis and regulation of phytoalexin coumarin were effectively activated. F6'H1 and S8H, encoding key enzymes in the biosynthesis of coumarins and their derivatives, were more strongly expressed at the late infection stage than at the early infection stage. Collectively, our study profiled the response pattern of citrus leaves against D. citri infection and provided the transcriptional evidence to support the defense mechanism.

The phyllosphere microbiome shifts toward combating melanose pathogen.

BACKGROUND: Plants can recruit beneficial microbes to enhance their ability to defend against pathogens. However, in contrast to the intensively studied roles of the rhizosphere microbiome in suppressing plant pathogens, the collective community-level change and effect of the phyllosphere microbiome in response to pathogen invasion remains largely elusive. RESULTS: Here, we integrated 16S metabarcoding, shotgun metagenomics and culture-dependent methods to systematically investigate the changes in phyllosphere microbiome between infected and uninfected citrus leaves by Diaporthe citri, a fungal pathogen causing melanose disease worldwide. Multiple microbiome features suggested a shift in phyllosphere microbiome upon D. citri infection, highlighted by the marked reduction of community evenness, the emergence of large numbers of new microbes, and the intense microbial network. We also identified the microbiome features from functional perspectives in infected leaves, such as enriched microbial functions for iron competition and potential antifungal traits, and enriched microbes with beneficial genomic characteristics. Glasshouse experiments demonstrated that several bacteria associated with the microbiome shift could positively affect plant performance under D. citri challenge, with reductions in disease index ranging from 65.7 to 88.4%. Among them, Pantoea asv90 and Methylobacterium asv41 identified as "recruited new microbes" in the infected leaves, exhibited antagonistic activities to D. citri both in vitro and in vivo, including inhibition of spore germination and/or mycelium growth. Sphingomonas spp. presented beneficial genomic characteristics and were found to be the main contributor for the functional enrichment of iron complex outer membrane receptor protein in the infected leaves. Moreover, Sphingomonas asv20 showed a stronger suppression ability against D. citri in iron-deficient conditions than iron-sufficient conditions, suggesting a role of iron competition during their antagonistic action. CONCLUSIONS: Overall, our study revealed how phyllosphere microbiomes differed between infected and uninfected citrus leaves by melanose pathogen, and identified potential mechanisms for how the observed microbiome shift might have helped plants cope with pathogen pressure. Our findings provide novel insights into understanding the roles of phyllosphere microbiome responses during pathogen challenge. Video abstract.

Abundant Genetic Diversity and Extensive Differentiation among Geographic Populations of the Citrus Pathogen Diaporthe citri in Southern China.

The fungal pathogen Diaporthe citri is a major cause of diseases in citrus. One common disease is melanose, responsible for large economic losses to the citrus fruit industry. However, very little is known about the epidemiology and genetic structure of D. citri. In this study, we analyzed 339 isolates from leaves and fruits with melanose symptoms from five provinces in southern China at 14 polymorphic simple sequence repeat (SSR) loci and the mating type idiomorphs. The genetic variations were analyzed at three levels with separate samples: among provinces, among orchards within one county, and among trees within one orchard. The five provincial populations from Fujian, Zhejiang, Jiangxi, Hunan, and Guizhou were significantly differentiated, while limited differences were found among orchards from the same county or among trees from the same orchard. STRUCTURE analysis detected two genetic clusters in the total sample, with different provincial subpopulations showing different frequencies of isolates in these two clusters. Mantel analysis showed significant positive correlation between genetic and geographic distances, consistent with geographic separation as a significant barrier to gene flow in D. citri in China. High levels of genetic diversity were found within individual subpopulations at all three spatial scales of analyses. Interestingly, most subpopulations at all three spatial scales had the two mating types in similar frequencies and with alleles at the 14 SSR loci not significantly different from linkage equilibrium. Indeed, strains with different mating types and different multilocus genotypes were frequently isolated from the same leaves and fruits. The results indicate that sexual reproduction plays an important role in natural populations of D. citri in southern China and that its ascospores likely represent an important contributor to citrus disease.

The Genome Sequence of the Citrus Melanose Pathogen Diaporthe citri and Two Citrus-Related Diaporthe Species.

Melanose disease is one the most widely distributed and economically important fungal diseases of citrus worldwide. The causative agent is the filamentous fungus Diaporthe citri (syn. Phomopsis citri). Here, we report the genome assemblies of three strains of D. citri, namely strains ZJUD2, ZJUD14, and Q7, which were generated using a combination of PacBio Sequel long-read and Illumina paired-end sequencing data. The assembled genomes of D. citri ranged from 52.06 to 63.61 Mb in genome size, containing 15,977 to 16,622 protein-coding genes. We also sequenced and annotated the genome sequences of two citrus-related Diaporthe species, D. citriasiana and D. citrichinensis. In addition, a database for citrus-related Diaporthe genomes was established to provide a public platform to access genome sequences, genome annotation and comparative genomics data of these Diaporthe species. The described genome sequences and the citrus-related Diaporthe genomes database provide a useful resource for the study of fungal biology, pathogen-host interaction, molecular diagnostic marker development, and population genomic analyses of Diaporthe species. The database will be updated regularly when the genomes of newly isolated Diaporthe species are sequenced. The citrus-related Diaporthe genomes database is freely available for nonprofit use at zjudata.com/blast/diaporthe.php.

Microbial communities and soil chemical features associated with commercial production of the medicinal mushroom Ganoderma lingzhi in soil.

Crop production, including mushroom farming, may cause significant changes to the underlying substrates which in turn, can influence crop quality and quantity during subsequent years. Here in this study, we analyzed the production of the medicinal mushroom Ganoderma lingzhi and the associated soil microbial communities and soil chemical features over 24 months from April 2015 to April 2017. This Basidiomycete mushroom, known as Lingzhi in China, is commonly found on dead trees and wood logs in temperate and subtropical forests. Its economic and medicinal importance have propelled the development of a diversity of cultivation methods. The dominant method uses wood logs as the main substrate, which after colonization by Lingzhi mycelia, are buried in the soil to induce fruiting. The soil microbial communities over the 24 months were analyzed using the Illumina HiSeq platform targeting a portion of the bacterial 16S rRNA gene and the fungal internal transcribed spacer 1 (ITS1). Overall, a significant reduction of Lingzhi yield was observed over our experimentation period. Interestingly, temporal changes in soil microbial compositions were detected during the 24 months, with the fungal community showing more changes than that of bacteria in terms of both species richness and the relative abundance of several dominant species after each fruiting. The soil chemical features also showed significant changes, with decreasing soil nitrogen and phosphorus concentrations and increasing soil pH and iron content after each fruiting. We discuss the implications of our results in sustainable Lingzhi production in soil.

Metabarcoding reveals differences in fungal communities between unflooded versus tidal flat soil in coastal saline ecosystem.

In the saline-affected ecosystem, fungi have huge potential to promote growth, induce disease resistance and enhance tolerance against salt-stress of host plants. Since areas of plowland are gradually decreasing, the reclamation of coastal saline lands could play a crucial role in maintaining agricultural productivity and crop security globally. Therefore, it is of great significance to explore the fungal diversity in the coastal saline ecosystem. Here, we collected saline soil samples from unflooded areas and tidal flat areas, the two typical distinct landforms in coastal saline ecosystems, and used ITS metabarcoding to depict the diversity of fungal communities. We found that fungal species evenness had a remarkably higher variation from the tidal flat compared to unflooded soil samples. Furthermore, we also confirmed that the fungal niches differentiation reports in the coastal saline ecosystem. Our ITS based DNA sequencing revealed that both unflooded and tidal flat soil were mainly composed of amplicon sequence variants (ASVs) belonging to Ascomycota (93.43% and 86.91% respectively). Based on our findings, understanding the associations and distinctions of fungal microbiome between unflooded soil and tidal flat could provide the basis for the development of reclamation in coastal saline lands.