Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering.

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious economic losses worldwide. For example, the citrus tristeza virus killed over 100 million citrus trees globally, while "Candidatus Liberibacter asiaticus" has cost Florida $9 billion. The use of pathogen-tested citrus budwood for tree propagation is key for the management of such pathogens. The Citrus Clonal Protection Program (CCPP) at the University of California, Riverside, uses polymerase chain reaction (PCR) assays to test thousands of samples from citrus budwood source trees every year to protect California's citrus and to provide clean propagation units to the National Clean Plant Network. A severe bottleneck in the high-throughput molecular detection of citrus viruses and viroids is the plant tissue processing step. Proper tissue preparation is critical for the extraction of quality nucleic acids and downstream use in PCR assays. Plant tissue chopping, weighing, freeze-drying, grinding, and centrifugation at low temperatures to avoid nucleic acid degradation is time-intensive and labor-intensive and requires expensive and specialized laboratory equipment. This paper presents the validation of a specialized instrument engineered to rapidly process phloem-rich bark tissues from citrus budwood, named the budwood tissue extractor (BTE). The BTE increases sample throughput by 100% compared to current methods. In addition, it decreases labor and the cost of equipment. In this work, the BTE samples had a DNA yield (80.25 ng/µL) that was comparable with the CCPP's hand-chopping protocol (77.84 ng/µL). This instrument and the rapid plant tissue processing protocol can benefit several citrus diagnostic laboratories and programs in California and become a model system for tissue processing for other woody perennial crops worldwide.

High-Throughput RNA Extraction from Citrus Tissues for the Detection of Viroids.

High-throughput nucleic acid extraction is critical for the implementation of modern viroid detection assays. Successful large-scale nursery, field surveys, and other regulatory, quarantine, or research diagnostic programs are increasingly dependent on high-throughput tissue pulverization and nucleic acid extraction protocols. Magnetic bead-based approaches using semi-automated robotic equipment allow high-throughput extraction and purification of high-quality uniform total nucleic acids for each individual sample. Here, we describe a streamlined and optimized protocol for citrus tissue processing and RNA extraction that can be used for downstream applications such as viroid detection by reverse transcription-quantitative polymerase chain reaction.

Genome analysis of Spiroplasma citri strains from different host plants and its leafhopper vectors.

BACKGROUND: Spiroplasma citri comprises a bacterial complex that cause diseases in citrus, horseradish, carrot, sesame, and also infects a wide array of ornamental and weed species. S. citri is transmitted in a persistent propagative manner by the beet leafhopper, Neoaliturus tenellus in North America and Circulifer haematoceps in the Mediterranean region. Leafhopper transmission and the pathogen's wide host range serve as drivers of genetic diversity. This diversity was examined in silico by comparing the genome sequences of seven S. citri strains from the United States (BR12, CC-2, C5, C189, LB 319, BLH-13, and BLH-MB) collected from different hosts and times with other publicly available spiroplasmas. RESULTS: Phylogenetic analysis using 16S rRNA sequences from 39 spiroplasmas obtained from NCBI database showed that S. citri strains, along with S. kunkelii and S. phoeniceum, two other plant pathogenic spiroplasmas, formed a monophyletic group. To refine genetic relationships among S. citri strains, phylogenetic analyses with 863 core orthologous sequences were performed. Strains that clustered together were: CC-2 and C5; C189 and R8-A2; BR12, BLH-MB, BLH-13 and LB 319. Strain GII3-3X remained in a separate branch. Sequence rearrangements were observed among S. citri strains, predominantly in the center of the chromosome. One to nine plasmids were identified in the seven S. citri strains analyzed in this study. Plasmids were most abundant in strains isolated from the beet leafhopper, followed by strains from carrot, Chinese cabbage, horseradish, and citrus, respectively. All these S. citri strains contained one plasmid with high similarity to plasmid pSci6 from S. citri strain GII3-3X which is known to confer insect transmissibility. Additionally, 17 to 25 prophage-like elements were identified in these genomes, which may promote rearrangements and contribute to repetitive regions. CONCLUSIONS: The genome of seven S. citri strains were found to contain a single circularized chromosome, ranging from 1.58 Mbp to 1.74 Mbp and 1597-2232 protein-coding genes. These strains possessed a plasmid similar to pSci6 from the GII3-3X strain associated with leafhopper transmission. Prophage sequences found in the S. citri genomes may contribute to the extension of its host range. These findings increase our understanding of S. citri genetic diversity.

Whole genome sequence of five strains of Spiroplasma citri isolated from different host plants and its leafhopper vector.

OBJECTIVES: Spiroplasma citri is a bacterium with a wide host range and is the causal agent of citrus stubborn and brittle root diseases of citrus and horseradish, respectively. S. citri is transmitted in a circulative, persistent manner by the beet leafhopper, Neoaliturus (Circulifer) tenellus (Baker), in North America. Five strains of S. citri were cultured from citrus, horseradish, and N. tenellus from different habitats and times. DNA from cultures were sequenced and genome assembled to expand the database to improve detection assays and better understand its genetics and evolution. DATA DESCRIPTION: The whole genome sequence of five strains of S. citri are described herein. The S. citri chromosome was circularized for all five strains and ranged from 1,576,550 to 1,742,208 bp with a G + C content of 25.4-25.6%. Characterization of extrachromosomal DNAs resulted in identification of one or two plasmids, with a G + C content of 23.3 to 27.6%, from plant hosts; and eight or nine plasmids, with a G + C content of 21.65 to 29.19%, from N. tenellus. Total genome size ranged from 1,611,714 to 1,832,173 bp from plants and 1,968,976 to 2,155,613 bp from the leafhopper. All sequence data has been deposited in DDBJ/ENA/GenBank under the accession numbers CP046368-CP046373 and CP047426-CP047446.

Genome Sequence Resource for Spiroplasma citri, Strain CC-2, Associated with Citrus Stubborn Disease in California.

Spiroplasma citri is a bacterium that causes stubborn disease of citrus and infects other crops, ornamentals, and weeds. It is transmitted by leafhoppers in a circulative manner. Due to limited sequence data on S. citri, the bacterium was isolated from naturally infected Chinese cabbage grown on a farm in Fresno County, CA. DNA from S. citri CC-2 was extracted from a pure culture in LD8 and subjected to PacBio sequencing. Four contigs were obtained with a single circular chromosome of 1,709,192 bp and three plasmids of 40,210, 39,313, and 2,921 bp in size. The genome developed herein extends the sequence database of S. citri and is the first whole-genome sequence record of S. citri from California.

An effector from the Huanglongbing-associated pathogen targets citrus proteases.

The citrus industry is facing an unprecedented challenge from Huanglongbing (HLB). All cultivars can be affected by the HLB-associated bacterium 'Candidatus Liberibacter asiaticus' (CLas) and there is no known resistance. Insight into HLB pathogenesis is urgently needed in order to develop effective management strategies. Here, we use Sec-delivered effector 1 (SDE1), which is conserved in all CLas isolates, as a molecular probe to understand CLas virulence. We show that SDE1 directly interacts with citrus papain-like cysteine proteases (PLCPs) and inhibits protease activity. PLCPs are defense-inducible and exhibit increased protein accumulation in CLas-infected trees, suggesting a role in citrus defense responses. We analyzed PLCP activity in field samples, revealing specific members that increase in abundance but remain unchanged in activity during infection. SDE1-expressing transgenic citrus also exhibit reduced PLCP activity. These data demonstrate that SDE1 inhibits citrus PLCPs, which are immune-related proteases that enhance defense responses in plants.

Genetic Structure of the Rice Blast Pathogen (Magnaporthe oryzae) over a Decade in North Central California Rice Fields.

Rice blast, caused by the ascomycete Magnaporthe oryzae, is one of the most destructive rice diseases worldwide. Even though the disease has been present in California since 1996, there is no data for the pathogen population biology in the state. Using amplified fragment length polymorphisms and mating-type markers, the M. oryzae population diversity was investigated using isolates collected when the disease was first established in California and isolates collected a decade later. While in the 1990 samples, a single multilocus genotype (MLG) was identified (MLG1), over a decade later, we found 14 additional MLGs in the 2000 isolates. Some of these MLGs were found to infect the only rice blast-resistant cultivar (M-208) available for commercial production in California. The same samples also had a significant decrease of MLG1. MLG1 was found infecting the resistant rice cultivar M-208 on one occasion whereas MLG7 was the most common genotype infecting the M-208. MLG7 was identified in the 2000 samples, and it was not present in the M. oryzae population a decade earlier. Our results demonstrate a significant increase in genotypic diversity over time with no evidence of sexual reproduction and suggest a recent introduction of new virulent race(s) of the pathogen. In addition, our data could provide information regarding the durability of the Pi-z resistance gene of the M-208. This information will be critical to plant breeders in developing strategies for deployment of other rice blast resistance genes/cultivars in the future.

A Pathogen Secreted Protein as a Detection Marker for Citrus Huanglongbing.

The citrus industry is facing an unprecedented crisis due to Huanglongbing (HLB, aka citrus greening disease), a bacterial disease associated with the pathogen Candidatus Liberibacter asiaticus (CLas) that affects all commercial varieties. Transmitted by the Asian citrus psyllid (ACP), CLas colonizes citrus phloem, leading to reduced yield and fruit quality, and eventually tree decline and death. Since adequate curative measures are not available, a key step in HLB management is to restrict the spread of the disease by identifying infected trees and removing them in a timely manner. However, uneven distribution of CLas cells in infected trees and the long latency for disease symptom development makes sampling of trees for CLas detection challenging. Here, we report that a CLas secreted protein can be used as a biomarker for detecting HLB infected citrus. Proteins secreted from CLas cells can presumably move along the phloem, beyond the site of ACP inoculation and CLas colonized plant cells, thereby increasing the chance of detecting infected trees. We generated a polyclonal antibody that effectively binds to the secreted protein and developed serological assays that can successfully detect CLas infection. This work demonstrates that antibody-based diagnosis using a CLas secreted protein as the detection marker for infected trees offers a high-throughput and economic approach that complements the approved quantitative polymerase chain reaction-based methods to enhance HLB management programs.

Novel diagnosis for citrus stubborn disease by detection of a spiroplasma citri-secreted protein.

Citrus stubborn disease (CSD), first identified in California, is a widespread bacterial disease found in most arid citrus-producing regions in the United States and the Mediterranean Region. The disease is caused by Spiroplasma citri, an insect-transmitted and phloem-colonizing bacterium. CSD causes significant tree damage resulting in loss of fruit production and quality. Detection of CSD is challenging due to low and fluctuating titer and sporadic distribution of the pathogen in infected trees. In this study, we report the development of a novel diagnostic method for CSD using an S. citri-secreted protein as the detection marker. Microbial pathogens secrete a variety of proteins during infection that can potentially disperse systemically in infected plants with the vascular flow. Therefore, their distribution may not be restricted to the pathogen infection sites and could be used as a biological marker for infection. Using mass spectrometry analysis, we identified a unique secreted protein from S. citri that is highly expressed in the presence of citrus phloem extract. ScCCPP1, an antibody generated against this protein, was able to distinguish S. citri-infected citrus and periwinkle from healthy plants. In addition, the antiserum could be used to detect CSD using a simple direct tissue print assay without the need for sample processing or specialized lab equipment and may be suitable for field surveys. This study provides proof of a novel concept of using pathogen-secreted protein as a marker for diagnosis of a citrus bacterial disease and can probably be applied to other plant diseases.

Development of molecular markers and preliminary investigation of the population structure and mating system in one lineage of black morel (Morchella elata) in the Pacific Northwestern USA.

Phylogenetic analysis of LSU/ITS sequence data revealed two distinct lineages among 44 morphologically similar fruiting bodies of natural black morels (Morchella elata group) sampled at three non-burn locations in the St Joe and Kanisku National Forests in northern Idaho. Most of the sampled isolates (n = 34) represented a dominant LSU/ITS haplotype present at all three sites and identical to the Mel-12 phylogenetic lineage (GU551425) identified in a previous study. Variation at 1-3 nucleotide sites was detected among a small number of isolates (n = 6) within this well supported clade (94%). Four isolates sampled from a single location were in a well supported clade (97%) distinct from the dominant haplotypes and may represent a previously un-sampled, cryptic phylogenetic species. Species-specific SNP and SCAR markers were developed for Mel-12 lineage isolates by cloning and sequencing AFLP amplicons, and segregation of AFLP markers were studied from single ascospore isolates from individual fruiting bodies. Based on the segregation of AFLP markers within single fruiting bodies, split decomposition analyses of two SCAR markers, and population genetic analyses of SNP, SCAR, and AFLP markers, it appears that members of the Morchella sp. Mel-12 phylogenetic lineage are heterothallic and outcross in nature similar to yellow morels. This is the first set of locus-specific molecular markers that has been developed for any Morchella species, to our knowledge. These markers will prove to be valuable tools to study mating system, gene flow and genetic structure of black morels at various spatial scales with field-collected fruiting bodies and eliminate the need to culture samples in vitro.