2021 FDA-NIH-NIST-USDA Joint Agency Microbiome (JAM) Symposium

Citrus Huanglongbing (HLB; yellow shoot disease) is associated with an unculturable a-proteobacterium “Candidatus Liberibacter asiaticus” (CLas). HLB was found in southern California in 2012, and the current management strategy is based on suppression of the Asian citrus psyllid (Diaphorina citri) that transmits CLas and removal of confirmed CLas-positive trees. Little is known about Asian citrus psyllid associated bacteria and citrus-associated bacteria in the HLB system. Such information is important in HLB management, particularly for accurate detection of CLas. Recent advancements in next-generation sequencing technology provide new opportunities to study HLB through genomic DNA sequence analyses (metagenomics). In this study, HLB-related bacteria in Asian citrus psyllid and citrus (represented by leaf midrib tissues) samples from southern California were analyzed. A metagenomic pipeline was developed to serve as a prototype for future bacteriomic research. This pipeline included steps of next-generation sequencing in Illumina platform, de novo assembly of Illumina reads, sequence classification using the Kaiju tool, acquisition of bacterial draft genome sequences, and taxonomic validation and diversity evaluation using average nucleotide identity. The identified bacteria in Asian citrus psyllids and citrus together included Bradyrhizobium, Buchnera, Burkholderia, “Candidatus Profftella armature,” “Candidatus Carsonella ruddii,” CLas, Mesorhizobium, Paraburkholderia, Pseudomonas, and Wolbachia. The whole genome of a CLas strain recently found in San Bernardino County was sequenced and classified into prophage typing group 1 (PTG-1), one of the five known CLas groups in California. Based on sequence similarity, Bradyrhizobium and Mesorhizobium were identified as possible source that could interfere with CLas detection using the 16S rRNA gene-based PCR commonly used for HLB diagnosis, particularly at low or zero CLas titer situation.

Jianchi Chen <jianchi.chen@usda.gov>

USDA-ARS

The microbiome of insects strongly influences many aspects of physiology and biology. In vector species, the microbial interactions also can influence the capacity of pathogen acquisition and transmission. Therefore, to better understand these associations that affect transmission success we selected a ‘non-vector’ psyllid specie in Florida to conduct comparative RNA-seq analyses of the microbiome: The wild lime psyllid, WLP, Leuronota fagarae Burckhardt (Hemiptera: Triozidae). Data analyses used in silico bioinformatics to putatively identify bacterial species in WLP and compare the presence and titers of these to published data for the microbiome of the Asian citrus psyllid, ACP adults. In ACP it has been shown that the microbiota shifts during the development, enabling acquisition of the pathogenic CLas in the first instars. Reduction of P. armatura was correlated with an increase CLas titers in ACP. The inverse was also reported with decreased transmission when P. armature are at increased titers over CLas. However, when Wolbachia-Dc titers in ACP are increased there is an increase in CLas transmission. Examination of the L. fagarae microbiome (extracted from 100 adults, both genders) showed less than 0.1% returns for Wolbachia, with close similarity to Wolb-pipiensis, not to Wolb-Dc in ACP. A novel Pantoea specie was identify (tentative named Pantoea leuronota). There are reports of Pantoea sp. in ACP and Potato psyllid . No 'Profftella' related endosymbiont was detected in WLP, However two unique clades to potential endosymbionts include Moraxella spp. (reported in aphids); and interestingly an endosymbiont related to a member in the Alphaproteobacteria (Candidatus Hodgkinia cicadicola) reported from Cicadas. Further analyses will evaluate potential roles in immunity and pathogen transmission.

Wayne Hunter <Wayne.hunter@usda.gov>

1.USDA, ARS, U.S. Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, Florida 34945. USA. Wayne.hunter@usda.gov; 2.ORISE participant, DOE/USDA, ARS, Fort Pierce, FL 34945, USA. Email: douglas.stuehler@gmail.com; 3. University of Florida, Southwest Florida Research and Education Center (SWFREC), 2685 SR 29 North Immokalee, Florida 34142. USA. jawwadq@ufl.edu; 4. Boyce Thompson Institute, Ithaca, NY 14853, USA; *Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, AZ 85721,USA; 5. University of Florida, IFAS, Dept. Plant Pathology, (IRREC), 2199 South Rock Rd, Ft. Pierce, FL 34945, USA. Email: lmcano@ufl.edu

Managing the soil microbiome using organic amendments is a promising approach for improving soil and crop health. Despite the growing interest in organic amendments, their effect on the soil microbiome is poorly characterized. This research tested the effect of green manure organic soil amendments on the taxonomic and functional variation of bacteria in the soil microbiome. Green manure amendments were generated from biomass of broccoli, marigold and sudangrass. The three green manures were amended to field soil and fallow (i.e., non-amended) soil was used as a negative control. Lettuce seedlings were transplanted into amended and fallow soil and maintained for twelve weeks under controlled conditions. Bacterial communities were characterized from soil DNA using nanopore sequencing of 16S rRNA and shotgun metagenome libraries. 16S and shotgun metagenome data were classified to bacterial genera using Kraken2. Shotgun metagenome data were also classified to functional hierarchies using MEGAN. Both 16S and shotgun metagenome data showed the taxonomic composition of bacterial communities was differentiated among the three types of green manures and the fallow negative control. Functional data showed more than one green manure amendment increased the abundance of bacterial traits related to iron and carbon acquisition and decreased bacterial traits related to type II and type IV secretion systems. This work demonstrates different green manure organic soil amendments can have specific effects on the taxonomic composition of bacterial communities, but may have overlapping effects on bacterial functional traits in the soil microbiome.

Nicholas LeBlanc

USDA-ARS

Soil health is an increasingly important concept that provides aspirational targets for agricultural management. Due in part to the incredible diversity of soil microbial communities, however, the biological components of soil health remain poorly understood. In this work we employ high-throughput sequencing of bacterial and fungal marker genes to evaluate soil microbial communities in relation to soil chemical parameters, biological soil health tests, and crop yields in fields under long-term reduced tillage (business-as-usual) and no tillage (aspirational) management at two soil depths. We found that tillage systems and soil depth were crucial determinants of microbial communities in conjunction with soil chemical measures. Moreover, although some measures of biological soil health (CO2-burst, Haney Soil Health Score) did not differentiate fields under different tillage systems, there were clear differences in microbial communities. The relative abundances of many microbial taxa were significantly related to soil chemical variables and crop yields. Fungal communities showed stronger correlations with yield than bacterial communities. Fungi from the families Sordariaceae, Hydnodontaceae, Hypocreaceae, and Clavicipitaceae were positively correlated with yield, especially in the upper soil depth, while Glomeraceae and Phaeosphaeriaceae were negatively correlated. Many of these correlations were also seen in the subsequent winter wheat and chickpea crops, and some correlations could be detected in the 20-year previous yield history of the no-tillage farm. Among the bacteria, only Microbaceriaceae and Xanthomonadaceae were positively correlated, but Caulobacteriaceae, Flavobacteriaceae, Sphingobacteriaceae, Chitinophagaceae were more abundant in lower yielding locations. This may reflect the strong plant selection in the rhizosphere for these bacterial groups in stressed plants. There were no significant differences between microbial diversity and management practices or significant correlations with crop yields. These results suggest that some specific groups of microbial taxa are responsive to tillage systems, exhibit positive relationships with grain yields, and may be useful indicators of soil health.

Dan Schlattter <daniel.schlatter@usda.gov>

USDA-ARS

Tar Spot is a foliar disease of corn, caused by the obligate biotrophic pathogen Phyllachora maydis (P. maydis), that has recently emerged as an economic concern for corn production in the United States. We investigated leaf microbiomes in relation to corn cultivars with differential tar spot symptoms under P. maydis infestation in natural field conditions. Leaf samples from sixteen corn cultivars were assessed for tar spot development and both bacterial and fungal microbiomes were characterized by paired-end sequencing on the Illumina MiSeq platform. Comparative metagenomics analysis of the leaf microbiomes revealed clear differences in overall microbial communities between resistant and susceptible corn cultivars. Bacterial communities in both resistant and susceptible lines were dominated by a single operational taxonomic unit (OTU) identified as Methylobacterium spp. The least-abundant OTU in resistant lines was in the genus Pseudomonas, whereas species of Microbacterium and Deinococcus were the least-abundant OTUs in susceptible lines. For fungal communities, genera Fusarium, Genolevuria, Zymoseptoria and Colletotrichum were least abundant whereas Papiliotrema and Alternaria were the most abundant OTUs in the resistant lines. By contrast, fungal communities in susceptible lines were dominated by two OTUs in the genera Puccinia and Sphaerellopsis. Further analyses of the differences in microbiome composition between susceptible and resistant cultivars could lead to the development of novel and more effective biological control agents against Tar Spot.

Raksha Singh

Crop Production and Pest Control Research Unit, U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS), Purdue University campus, West Lafayette, IN 47907