Host Plant DNA Research Articles

Southern blight of water lily: The first host record of Agroathelia rolfsii on Nelumbo nucifera discovered in Florida, USA.

Nelumbo nucifera Gaertn. (Nelumbonaceae, Eudicots), also known as water lily or sacred lotus, is a nonnative and invasive plant commonly found in artificial ponds and natural lakes throughout Florida (UF-IFAS 2023; Wunderlin et al. 2023). In August 2020, a single sample of water lily plants showing large leaf spots were collected at a residence in Dunnellon, Marion County, Florida (80% disease prevalence with 40% leaf coverage). Symptoms and signs of the disease were necrotized adaxial leaf spots only, bordered by whitish mycelia and hyphae with clamp connections, and whitish to light brown sclerotia formed in the center (<0.7 mm diameter). Symptomatic tissue was plated on acid potato dextrose agar (APDA) amended with chloramphenicol (100 mg/L) and ampicillin (30mg/L), and incubated at 20 °C for one week. Data supporting the molecular identification of this putative pathogen were gathered by PCR amplification and Sanger sequencing of the complete internal transcribed spacer (ITS) and a fragment of the large subunit (LSU) of the rRNA gene (~1.5 kb) using primers ITS1F and LR5 (FDACS-DPI PPST 2020-105211, GenBank OR492009) (White et al. 1990). The identification of the host was confirmed by Sanger sequencing of three plant barcode fragments: ITS2 (ITS2-S2F/ITS4, OR492008), ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) (rbcLa-F/rbcLa-R, GenBank OR502388), and Maturase K (matK) (matK-KIM1R/matK-KIM3F, GenBank OR502389) (Fazekas et al. 2012). MegaBLAST queries of the ITS/LSU sequence obtained here recovered a 99.61% match to the fungal pathogen Agroathelia (=Athelia) rolfsii (Sacc.) Redhead & Mullineux. (Redhead and Mullineux 2023) (Amylocorticiaceae, Agaricomycotina) strain GP3 (GenBank JABRWF010000005) (Yan et al. 2021). MegaBLAST queries of three host plant DNA barcodes recovered matches of greater than 99.62% similarity to N. nucifera sequences. After diagnosis, symptomatic dried leaf samples were deposited at Plant Industry Herbarium Gainesville (PIHG 17807) and an axenic culture was deposited at the Agricultural Research Services Culture Collection (NRRL 66964). Koch's postulates were fulfilled by the inoculation of sclerotia (as in Terrones-Salgado et al. 2022) on adaxial leaf surface of four-week- old water lily transplants obtained from an artificial pond on campus (two plants with five leaves each). One additional transplant was not inoculated and served as a control; this plant remained asymptomatic during the experimentation period. Each transplant was kept in a 27-gallon plastic container (21W × 30L × 14H in) filled with tap water containing one tablespoon of 20-20-20 all-purpose-water-soluble plant fertilizer (VPG, TX, USA) in a plant biosafety level 2 greenhouse (23 °C, >50% relative humidity, and a 12-h/12-h photoperiod). All inoculated leaves showed necrotized areas after one week and new sclerotia were observed floating on the water surface after three weeks. Fungal pathogen was reisolated and reidentified subsequently. Agroathelia rolfsii is the causal agent of southern blight, also known as grey rot, and is reported from at least in 260 plant genera, including specialty crops such as citrus, cucumber, pepper, peanuts, pumpkin, and strawberry (Farr and Rossman 2018). Agroathelia rolfsii usually causes lower stem, crown, and root rots; consequently, leaf spots are a noteworthy presentation of symptoms for this fungus.

Chip Digital PCR (cdPCR) to Identify and Quantify Botrytis cinerea Infection in Tomatoes

Botrytis cinerea is a fungal pathogen present in almost any environment, able to cause a severe postharvest disease on a wide range of crops, resulting in significant economic losses. Furthermore, B. cinerea is frequently found in plant tissues as a latent, asymptomatic infection that, when stimulated by favorable alterations in the environment or the physiology of the host, can swiftly develop into a significant symptomatic infection. In greenhouses, fields, and on propagation materials, the principal strategy adopted to control infection is the use of chemical fungicides or eco-friendly alternative methods. For the optimal success of conventional and biocontrol treatments, it is crucial to monitor the disease development and the fungal infection entity. The aim of this work was to develop a fast new method based on chip digital PCR (cdPCR) to estimate the extent of the B. cinerea infection in tomatoes. To better evaluate the amount of plant infection, a duplex assay able to co-amplify both fungal and host plant DNA was fine-tuned. The cdPCR assays were applied to quantify B. cinerea in tomato seedling samples, both naturally and artificially contaminated. The developed method offers sensitive detection, reliable identification, and precise pathogen quantification. The method can be used for B. cinerea diagnostics along the tomato production chain, starting from the seeds and transplanting seedlings to plants and crop residues in open fields and greenhouses. To the best of our knowledge, this is the first study directed at applying cdPCR to B. cinerea diagnosis in tomatoes.

The aphid Myzus persicae (Hemiptera: Aphididae) acquires chloroplast DNA during feeding on host plants.

Aphids (Hemiptera: Aphididae) extract nutrients from host plant phloem via stylets that facilitate salivation and sap uptake. When navigating to the phloem, aphids periodically puncture nonvascular cells and sample cell contents, but rarely cause significant cell damage. As a result, aphids are considered "stealthy" feeders. In contrast, insects that do cause damage, such as chewing herbivores, will take up host cell contents-including DNA-into their guts. Researchers can use molecular barcoding methods to identify recent host use patterns of chewing herbivores. This information is valuable for both pest management and basic ecological studies. Because of their stealthy feeding style, it was assumed that host plant DNA could not be recovered from aphids and other Sternorrhyncha. However, several recent studies document host plant DNA uptake by psyllids, which feed in a similar manner to aphids. Therefore, we hypothesized that aphids may also acquire DNA from host plants. Since aphids puncture and sample cytosol contents from cells, we predicted that aphids would be most likely to acquire DNA from chloroplasts. To test this, we performed host feeding and host transfer experiments with Myzus persicae (Sulzer), then used PCR to recover and sequence a region between the trnT and trnF genes from acquired chloroplast DNA. We found that M. persicae readily acquires chloroplast DNA, even prior to phloem contact, and that fragment sizes sufficient for host plant identification can be recovered. Our work suggests that molecular gut content analysis is a viable tool for studying aphid-host interactions.

Does peptide-nucleic acid (PNA) clamping of host plant DNA benefit ITS1 amplicon-based characterization of the fungal endophyte community?

Fungal endophyte community amplicon sequencing can lose a significant number of informative reads due to host-plant co-amplification. Blocking of plant-specific sequences with peptide nucleic acid (PNA) clamps has been shown to improve metrics of detected microbial diversity in studies targeting 16S and 18S regions of rRNA genes. However, PNA clamping has not been applied to the plant ITS region of rRNA gene – a widely accepted fungal marker. By applying PNA clamping technique to ITS amplicon sequencing of the endophytic fungal community of elderberry this study shows that PNA clamping significantly reduces host-plant co-amplification with the universal ITS1/ITS4 primer set. However, PNA clamping in combination with the discriminatory ITS1F/ITS2 primer set did not improve the metrics of fungal endophyte community ITS amplicon Illumina sequencing. This study shows that PNA clamping does not add practical benefit to taxonomic profiling of plant-associated fungal communities if the primers are already specific enough to exclude amplification of host DNA. • PNA clamping of host-plant DNA can be applied to fungal marker locus ITS1 . • PNA clamping is beneficial in combination with universal ITS1/ITS4 primer set. • PNA clamping did not improve performance of specific Illumina ITS1F/ITS2 primer set. • Results challenge the practicality of PNA clamping for ITS Illumina sequencing.

New distribution records, first host plant record and DNA barcoding of the Neotropical plume moth Oidaematophoruspseudotrachyphloeus Gielis (Lepidoptera, Pterophoridae).

BackgroundOidaematophoruspseudotrachyphloeus Gielis, 2011 (Lepidoptera, Pterophoridae) is a little-known Neotropical plume moth previously recorded in Ecuador, Peru and Argentina. Its host plants and DNA barcodes are unknown.New informationOidaematophoruspseudotrachyphloeus is recorded for the first time from Chile, based on six specimens from the Azapa Valley (Arica Province) and two from Guayacán (Coquimbo Province). Those from the Azapa Valley were reared from folivorous larvae collected on Ambrosiacumanensis Kunth (Asteraceae), representing the first host plant record for this plume moth. The first DNA barcode sequences of O.pseudotrachyphloeus are provided and used to explore relationships with congenerics.

Host Plant Signal Persistence in the Gut of the Brown Marmorated Stink Bug (Hemiptera: Pentatomidae).

Determining the host range of an invasive insect in a new environment is a key step in the development of management strategies. As the brown marmorated stink bug, Halyomorpha halys Stål, expands into agricultural regions of North America, efforts to elucidate its dietary habits on a landscape scale rely on intensive sampling of potential host plants. Although this approach yields useful information, results can be biased toward common and easily sampled plant species; important hosts can be missed if sampling them is impractical or limited in scope. Here we lay the groundwork for the application of gut content analysis to the feeding ecology of H. halys by investigating the persistence of host plant DNA in the digestive tracts of insects with known feeding histories. Adult H. halys were fed bean seedlings (Phaseolus lunatus L.) for 7 d, followed by a forced host switch to carrot (Daucus carota L.). Insect guts were dissected out at 0, 1, 3, 7, and 14 d following the switch, and host plant chloroplast genes (trnF and trnL) were amplified via polymerase chain reaction. Amplicons were identified using high-throughput sequencing and analyzed for Phaseolus DNA. The original host remained detectable at 3 d (trnF) and 14 d (trnL) in substantial quantities. The proportion of total reads identified as Phaseolus rapidly decreased with time; a concomitant increase in Daucus reads was observed. Our results indicate that high-throughput sequencing of gut contents has great potential for exploring the dietary histories of field-caught H. halys and other phytophagous insects.

Detecting Ingested Host Plant DNA in Potato Leafhopper (Hemiptera: Cicadellidae): Potential Use of Molecular Markers for Gut Content Analysis.

Detection of host plant DNA from sap-feeding insects can be challenging due to potential low concentration of ingested plant DNA. Although a few previous studies have demonstrated the possibility of detecting various fragments of plant DNA from some sap-feeders, there are no protocols available for potato leafhopper, Empoasca fabae (Harris) (Hemiptera: Cicadellidae), a significant agricultural pest. In this study we focused on optimizing a DNA-based method for host plant identification of E. fabae and investigating the longevity of the ingested plant DNA as one of the potential applications of the protocol. We largely utilized and modified our previously developed PCR-based method for detecting host plant DNA from grasshopper and the spotted lanternfly gut contents. We have demonstrated that the trnL (UAA) gene can be successfully utilized for detecting ingested host plant DNA from E. fabae and determining plant DNA longevity. The developed protocol is a relatively quick and low-cost method for detecting plant DNA from E. fabae. It has a number of important applications-from determining host plants and dispersal of E. fabae to developing effective pest management strategies.

Event-Specific Qualitative and Quantitative Detection of Genetically Modified Rice G6H1

The genetically modified rice G6H1 expressing a fused protein of Cry1Ab/Vip3H and a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) G6 is a genetically modified event that has been approved for preproduction field testing in China. The purpose of this study was to establish an event-specific qualitative and quantitative detection method that could provide a stable, reliable system for monitoring this new transgenic event. In this study, event-specific qualitative and quantitative detection methods based on the 3′ integration junction sequence between host plant DNA and the integrated gene were developed. The limit of detection (LOD) of qualitative PCR was assessed to be 0.1%. The LOD of quantitative PCR was estimated to be ten haploid genome copies. The quantitative PCR detection method was verified with three mixed rice samples with known G6H1 contents, and the results agreed with the expected values. Evaluation of specificity and sensitivity indicated that the developed qualitative and quantitative PCR methods are reliable and can be used for the detection and quantification of G6H1.

The specific host plant DNA detection suggests a potential migration of Apolygus lucorum from cotton to mungbean fields

The polyphagous mirid bug Apolygus lucorum (Heteroptera: Miridae) has more than 200 species of host plants and is an insect pest of important agricultural crops, including cotton (Gossypium hirsutum) and mungbean (Vigna radiata). Previous field trials have shown that A. lucorum adults prefer mungbean to cotton plants, indicating the considerable potential of mungbean as a trap crop in cotton fields. However, direct evidence supporting the migration of A. lucorum adults from cotton to mungbean is lacking. We developed a DNA-based polymerase chain reaction (PCR) approach to reveal the movement of A. lucorum between neighboring mungbean and cotton fields. Two pairs of PCR primers specific to cotton or mungbean were designed to target the trnL-trnF region of chloroplast DNA. Significant differences in the detectability half-life (DS50) were observed between these two host plants, and the mean for cotton (8.26 h) was approximately two times longer than that of mungbean (4.38 h), requiring weighted mean calculations to compare the detectability of plant DNA in the guts of field-collected bugs. In field trials, cotton DNA was detected in the guts of the adult A. lucorum individuals collected in mungbean plots, and the cotton DNA detection rate decreased successively from 5 to 15 m away from the mungbean-cotton midline. In addition to the specific detection of cotton- and mungbean-fed bugs, both cotton and mungbean DNA were simultaneously detected within the guts of single individuals caught from mungbean fields. This study successfully established a tool for molecular gut-content analyses and clearly demonstrated the movement of A. lucorum adults from cotton to neighboring mungbean fields, providing new insights into understanding the feeding characteristics and landscape-level ecology of A. lucorum under natural conditions.

Novel PCR Assays for the Detection of Biological Agents Responsible for Wheat Rust Diseases: Puccinia triticina and Puccinia striiformis f. sp. tritici

The species Puccinia triticina (Pt) and Puccinia striiformis f. sp. tritici (Pst) are devastating cereal pathogens that cause leaf and stripe rust diseases. We developed PCR assays for the species-specific detection of Pt and Pst, 2 biological agents that cause wheat rust disease. For each pathogen, we validated 3 primer sets that target the second largest subunits of the RNA polymerase II (rpb2) and β-tubulin 1 (tub1) genes. The specificities of the primers were verified using naturally infected plant materials with visual symptoms of disease. All primer sets amplified a single DNA fragment of the expected length. The primer sets LidPr15/16, LidPr1/2, and LidPs13/14 were able to detect small amounts of pure fungal DNA with sensitivities of 0.1, 1, and 10 pg/μL, respectively. A sufficient detection limit (1 pg/μL to 5 ng/μL) was observed for all assays when the sensitivity test was performed with host plant DNA. The study also evaluated the simultaneous detection of both rust pathogens, and the multiplex PCR assay generated amplicons of 240 and 144 bp in length for Pts (LidPs9/10) and Pt (LidPr1/2), respectively.

Analysis of endophytic actinobacteria species diversity in the stem of Gynura cusimbua by 16S rRNA gene clone library

Endophytic actinobacteria that lived in any associations with plant tissues represented a rather unexplored area of actinobacteria compared with soils. Gynura cusimbua was a kind of medicinal plant which had prevention effects for high blood pressure, coronary heart disease, Alzheimer’s disease, atherosclerosis, etc. Endophytic actinobacteria of G. cusimbua might produce some secondary metabolites which had the same function as their host. Stem samples of G. cusimbua collected from Hainan Province were used to study their endophytic actinobacteria to find some new compounds. In order to avoid vast proportions of the host plant DNA in the metagenomic library, the strategies of enrichment of the microorganism cells after tissue digestion and exclusion of 16S rRNA gene derived from the plastid by digested with PvuII were used. Two sets of actinobacteria specific primers were used for targeting endophytic actinobacteria from metagenomic library. 63 positive clones of actinobacteria were selected for sequencing and constructing the phylogenetic tree of 16S rRNA gene, and the 16S rRNA gene sequence of 59 strains among them had higher similar to the closest type strain and belonged to Microbacterium, Arthrobacter, Micrococcus, Curtobacterium, Okibacterium, Quadrisphaera and Kineococcus, respectively. Others were in low similarity and belonged to unclassified Micrococcineae, unclassified Intrasporangiaceae and unclassified Microbacteriaceae.

The Impact of Environmental Conditions on Efficiency of Host Plant DNA Barcoding for Polyphagous Beetles.

Recently, several papers were published dealing with host plant identification for selected species of insects, including beetles. These studies took advantage of the DNA barcoding approach and generally showed that it is possible to identify diet composition from plant DNA present in insect guts. However, none of these studies considered how the impact of environmental conditions affected the likelihood of insect feeding and, therefore, the presence of host plant DNA that could be amplified and sequenced. In the present study, individuals of the polyphagous weevil Centricnemus leucogrammus (Germar, 1824) (Curculionidae: Entiminae) were used to test the hypothesis that harsh environmental conditions limited its feeding activity. The diet of 50 specimens collected during favourable conditions in the middle of the species reproductive period was compared against the diet of 50 specimens collected during harsh environmental conditions. Results clearly showed that almost no weevils fed during rainy and cold conditions and only a minority of individuals (20%) fed during the drought condition (on drought-resistant plants). It is important to consider such factors in any studies dealing with host plant identification and feeding behaviour. Results of ecological studies could lead to erroneous conclusions, e.g., underestimation of number and composition of host plants in the diet of studies species.

Loop-Mediated Isothermal Amplification (LAMP) for Detection of Phytoplasmas in the Field.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid-based detection method with many applications. This chapter details its use for detection of phytoplasma diseases, combining a rapid 2-min DNA extraction method with real-time LAMP product detection such that the entire procedure can be undertaken and completed in the field within 40 min. Furthermore, the assays include an anneal curve validation step to guard against false positives and an assay for host plant DNA to guard against false negatives.

Microbial Pathogens Trigger Host DNA Double-Strand Breaks Whose Abundance Is Reduced by Plant Defense Responses

Immune responses and DNA damage repair are two fundamental processes that have been characterized extensively, but the links between them remain largely unknown. We report that multiple bacterial, fungal and oomycete plant pathogen species induce double-strand breaks (DSBs) in host plant DNA. DNA damage detected by histone γ-H2AX abundance or DNA comet assays arose hours before the disease-associated necrosis caused by virulent Pseudomonas syringae pv. tomato. Necrosis-inducing paraquat did not cause detectable DSBs at similar stages after application. Non-pathogenic E. coli and Pseudomonas fluorescens bacteria also did not induce DSBs. Elevation of reactive oxygen species (ROS) is common during plant immune responses, ROS are known DNA damaging agents, and the infection-induced host ROS burst has been implicated as a cause of host DNA damage in animal studies. However, we found that DSB formation in Arabidopsis in response to P. syringae infection still occurs in the absence of the infection-associated oxidative burst mediated by AtrbohD and AtrbohF. Plant MAMP receptor stimulation or application of defense-activating salicylic acid or jasmonic acid failed to induce a detectable level of DSBs in the absence of introduced pathogens, further suggesting that pathogen activities beyond host defense activation cause infection-induced DNA damage. The abundance of infection-induced DSBs was reduced by salicylic acid and NPR1-mediated defenses, and by certain R gene-mediated defenses. Infection-induced formation of γ-H2AX still occurred in Arabidopsis atr/atm double mutants, suggesting the presence of an alternative mediator of pathogen-induced H2AX phosphorylation. In summary, pathogenic microorganisms can induce plant DNA damage. Plant defense mechanisms help to suppress rather than promote this damage, thereby contributing to the maintenance of genome integrity in somatic tissues.

Development of an event-specific assay for the qualitative and quantitative detection of the genetically modified flax CDC Triffid (FP967)

The genetically modified flax, event FP967, with tolerance to sulfonylurea herbicides, is one of the commercial genetically modified events approved in Canada and USA.This event is not authorized in Switzerland and EU, therefore, a method to specifically detect the CDC Triffid line, was required. We revealed the 3′ integration junction sequence between host plant DNA and the integrated gene construct FP967 by means of Restriction Site PCR and both a qualitative and a quantitative PCR detection assays were developed. The qualitative PCR revealed a limit of detection of 0.01% of GM flax in 100 ng of genomic DNA. The quantitative PCR assay showed a limit of detection of about 9 haploid genome copies. The specificity and sensitivity of the assay indicate that the developed event-specific PCR methods can be used for identification and quantification of FP967 flax.

Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis

The xylem-limited, Gram-negative, fastidious plant bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease affecting approximately half of the citrus plantations in the State of São Paulo, Brazil. The disease was recently found in Central America and is threatening the multi-billion U.S. citrus industry. Many strains of X. fastidiosa are pathogens or endophytes in various plants growing in the U.S., and some strains cross infect several host plants. In this study, a TaqMan-based assay targeting the 16S rDNA signature region was developed for the identification of X. fastidiosa at the species level. Another TaqMan-based assay was developed for the specific identification of the CVC strains. Both new assays have been systematically validated in comparison with the primer/probe sets from four previously published assays on one platform and under similar PCR conditions, and shown to be superior. The species specific assay detected all X. fastidiosa strains and did not amplify any other citrus pathogen or endophyte tested. The CVC-specific assay detected all CVC strains but did not amplify any non-CVC X. fastidiosa nor any other citrus pathogen or endophyte evaluated. Both sets were multiplexed with a reliable internal control assay targeting host plant DNA, and their diagnostic specificity and sensitivity remained unchanged. This internal control provides quality assurance for DNA extraction, performance of PCR reagents, platforms and operators. The limit of detection for both assays was equivalent to 2 to 10 cells of X. fastidiosa per reaction for field citrus samples. Petioles and midribs of symptomatic leaves of sweet orange harbored the highest populations of X. fastidiosa, providing the best materials for detection of the pathogen. These new species specific assay will be invaluable for molecular identification of X. fastidiosa at the species level, and the CVC specific assay will be very powerful for the specific identification of X. fastidiosa strains that cause citrus variegated chlorosis.

Event-specific detection of genetically modified wheat B73-6-1 based on the 3′-flanking sequence

In this study, 3′-flanking sequence between the host plant DNA and the integrated gene construct of pHMW1Dx5 vector in transgenic wheat B73-6-1 was revealed by means of adaptor PCR; thus, the fragment with the length of 3.1 kb was obtained, including a 190-bp wheat genomic DNA, which demonstrates that this HMW-GS gene was located on the wheat chromosome 3B. And the event-specific PCR primers were designed based upon the revealed 3′-flanking sequence; the conventional qualitative PCR and quantitative SYBR real-time PCR detection methods employing these primers were successfully developed. In conventional qualitative PCR assay, the limit of detection was 0.1 % for B73-6-1 wheat genomic DNA for one reaction. In the quantitative SYBR real-time PCR assay, the limit of detection and limit of quantification were 10 and 100 haploid genome copies, respectively. In addition, three mixed blind wheat samples with known B73-6-1 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event-specific real-time PCR detection systems were reliable, sensitive and accurate.

Food assimilated by two sympatric populations of the brown planthopper Nilaparvata lugens (Delphacidae) feeding on different host plants contaminates insect DNA detected by RAPD-PCR analysis

Contamination of insect DNA for RAPD-PCR analysis can be a problem because many primers are non-specific and DNA from parasites or gut contents may be simultaneously extracted along with that of the insect. We measured the quantity of food ingested and assimilated by two sympatric populations of brown planthopper (BPH), Nilaparvata lugens, one from rice and the other from Leersia hexandra (Poaceae), a wetland forage grass, and we also investigated whether host plant DNA contaminates that of herbivore insects in extractions of whole insects. Ingestion and assimilation of food were reduced significantly when individuals derived from one host plant were caged on the other species. The bands, OPA3 (1.25), OPD3 (1.10), OPD3 (0.80), OPD3 (0.60), pUC/M13F (0.35), pUC/M13F (0.20), BOXAIR (0.50), peh#3 (0.50), and peh#3 (0.17) were found in both rice-infesting populations of brown planthopper and its host plant (rice). Similarly, the bands, OPA4 (1.00), OPB10 (0.70), OPD3 (0.90), OPD3 (0.80), OPD3 (0.60), pUC/ M13F (0.35), pUC/M13F (0.20), and BOXAIR (0.50) were found in both Leersia-infesting populations of brown planthopper and the host plant. So, it is clear that the DNA bands amplified in the host plants were also found in the extracts from the insects feeding on them.

Differential filtration approach for isolation and enrichment of Sugarcane Grassy Shoot phytoplasma

The phytoplasma-associated grassy shoot disease is one of the major threats in sugarcane cultivation. A rapid method for enrichment and isolation of Sugarcane Grassy Shoot (SCGS) phytoplasma from the infected plants was developed. Differential filtration approach was used to isolate and enrich the SCGS phytoplasma and its genomic DNA that was detected by PCR analysis for phytoplasmal 16S rDNA. Ratio of pathogen to host plant DNA was found in the order of 103 and 105 in infected tissue and enriched fraction respectively, offering 148-fold increase in sensitivity for their detection.

Enriching plant microbiota for a metagenomic library construction

Plant microbiota (the microorganisms that live in any associations with plant tissues) represents a rather unexplored area of metagenomic research compared with soils and oceans. Constructing a metagenomic library for plant microbiota is technically challenging. Using all the biomass without pre-enrichment could lead to vast proportions of the host plant DNA in the metagenomic library, doubtless obliterating the microbial contribution. Therefore, the first and essential step is to enrich for the constituent microorganisms from plant tissues. Here, a strong enrichment for plant microbiota was achieved by coupling SDS (sodium dodecyl sulfate) with NaCl, creating a predominantly microbial metagenomic library that contains 88% bacterial inserts. 16S rDNA sequence analysis revealed that the metagenomic DNA of enrichments originates from very diverse microorganisms. At least 74 distinct ribotypes (at a 97% threshold) from seven different bacterial phyla were identified and mainly distributed among Actinobacteria and Proteobacteria. Additionally, a simplified version of Amplified Ribosomal DNA Restriction Analysis (ARDRA) was developed for a quick and efficient assessment of the enriching procedures. This work opens further insight into the great biotechnical potential of plant microbiota, holding more potential for drug discovery through a metagenomic strategy, and paving the way for recovery and biochemical characterization of functional gene repertoire from plant microbiota.