Evidence of Xylella fastidiosa Infection and Associated Thermal Signatures in Southern Highbush Blueberry (Vaccinium corymbosum Interspecific Hybrids).

Xylella fastidiosa, a gram-negative bacterium vectored to plants via feeding of infected insects, causes a number of notorious plant diseases throughout the world, such as Pierce's disease (grapes), olive quick decline syndrome, and coffee leaf scorch. Detection of Xf in infected plants can be challenging because the early foliar disease symptoms are subtle and may be attributed to multiple minor physiological stresses and/or borderline nutrient deficiencies. Furthermore, Xf may reside within an infected plant for one or more growing seasons before traditional visible diagnostic disease symptoms emerge. Any method that can identify infection during the latent period or pre-diagnostic disease progress state could substantially improve the outcome of disease control interventions. Because Xf locally and gradually impairs water movement through infected plant stems and leaves over time, infected plants may not be able to effectively dissipate heat through transpiration-assisted cooling, and this heat signature may be an important pre-diagnostic disease trait. Here, we report on the association between thermal imaging, the early stages of Xf infection, and disease development in blueberry plants, and discuss the benefits and limitations of using thermal imaging to detect bacterial leaf scorch of blueberries.

Complete Genome Sequences of Xylella fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex Strains Causing Blueberry Bacterial Leaf Scorch Disease in Georgia, USA.

Here, we report the complete genome sequences of Xylella fastidiosa subsp. fastidiosa strain AlmaReb2 and X. fastidiosa subsp. multiplex strain AlmaRebR6, causing blueberry bacterial leaf scorch disease in Georgia, USA. The X. fastidiosa subsp. fastidiosa AlmaRebR2 chromosome is 2,549,422 bp, and the X. fastidiosa subsp. multiplex AlmaReb6 chromosome is 2,530,348 bp.

First Report of Bacterial Wilt Disease Caused by Ralstonia solanacearum on Southern Highbush Blueberries (Vaccinium corymbosum interspecific hybrids) in Georgia U.S.A.

Members of the Ralstonia solanacearum (Rs) species complex have recently been reported to cause bacterial wilt on southern highbush (SHB) blueberries in Florida (Norman et al. 2018), a disease first reported on blueberry (northern highbush; Vaccinium corymbosum) in New Jersey (Patel et al. 2013). SHB blueberries are widely grown in the southern United States, and SHB cultivars represent the majority of the blueberry acreage in Georgia - the U.S. state with the largest blueberry acreage (NASS 2022). In Fall 2020, three-year old SHB plants (cv. 'Indigocrisp') showing leaf bronzing, wilting, and dieback were collected from two field sites in Clinch County, GA. At these locations, numerous plants were rapidly dying, with symptoms appearing to have spread down rows. Plant material tested positive using the ImmunoStrip® for Rs (Agdia, Inc., Elkhart, IN). From one location, the remaining sample was submitted to USDA-APHIS Select Agent Services who determined that Rs was present but a select agent (Rs Race 3, Biovar 2) was not. Following this, six adjacent, symptomatic SHB plants were collected from the same field location. These plants tested positive using the ImmunoStrip® for Rs, and red-pigmented mucoid colonies typical of Rs formed within 48 h at 28°C on triphenyltetrazolium chloroide (TZC) isolation medium (Kelman 1954). DNA was extracted from pure Rs cultures using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1987) and tested using polymerase chain reaction (PCR). Primers pairs AMB013/AMB014 (Fegan and Prior 2005) and ENDO-F/ENDO-R (Ji et al. 2007) were used to amplify 558 nt and 843 nt portions of the 16S rRNA region and Rs endoglucanase gene, respectively. Resulting amplicons were purified using an E.Z.N.A.® Cycle Pure Kit (Omega Bio-Tek, Norcross, GA), Sanger sequenced in both directions (Eurofins Genomics, Louisville, KY), and compared to publicly available Rs sequences in Genbank. The 16 rRNA sequence from all obtained isolates (accession ON938207) had 100% identity to Rs strain CFBP2957 (FP885897), while the endoglucase sequence (ON938206) had 100% identity to phylotype IIa, sequevar 5 Rs strain CIP-426 (MF461810) and phylotype IIa, sequevar 39 Rs strain 19-058 (MT314067), among others. To fulfill Koch's postulates, an isolate ('Ral21-1') was grown on TZC medium for 48 h at 28°C and suspended in 8.5 g/L NaCl at 1 x 108 CFU/ml. Five young, tissue cultured SHB plants (cv. 'Kestrel') in 25 cm pots were drenched with 50 ml of Rs suspension. For six weeks, plants were maintained in the greenhouse at 21-32°C. Typical bacterial wilt symptoms (leaf bronzing/scorching) developed in all inoculated plants, and infections were confirmed using Immunostrip®. Rs was reisolated and confirmed via PCR and sequencing as previously described. While Ralstonia has been known to cause disease on numerous crops in Georgia, this represents a first report of bacterial wilt in Georgia blueberries. Relative to rabbiteye blueberries (V. virgatum), recent reports suggest that SHB are much more susceptible to bacterial wilt (Conner et al. 2022). Accordingly, given the transition from rabbiteye to SHB within Georgia's blueberry production region over the past two decades and the ability of Rs to spread easily in water, soil, or via infected plant material, the presence of this disease within the state represents a significant threat to blueberry production. Additional characterization of Ralstonia isolates from Georgia may help assess the risk of future outbreaks.

Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry.

Bacterial leaf scorch (BLS), caused by Xylella fastidiosa (Xf), is a prevalent disease of blueberries in the southeastern United States. Initially, this disease was reported to be caused by X. fastidiosa subsp. multiplex (Xfm). However, a recent survey revealed the presence of another subspecies, X. fastidiosa subsp. fastidiosa (Xff), within naturally infected blueberry plantings in Georgia. Since knowledge regarding the origins of isolates causing Xf outbreaks can impact management recommendations, a routine method for identifying the pathogen at the subspecies level can be beneficial. Several detection strategies are available to identify Xf infection at the subspecies level. However, none of these have been developed for the routine and rapid differentiation of the blueberry-infecting Xf subspecies. Here, we developed two separate straightforward and rapid detection techniques, a cleaved amplified polymorphic sequence (CAPS) marker, and a loop-mediated isothermal amplification (LAMP) assay, targeting the RNA polymerase sigma-70 factor (rpoD) gene sequence of Xfm to discriminate between the two Xf subspecies infecting blueberry. With the CAPS marker, specific detection of Xfm isolates was possible from pure cultures, inoculated greenhouse-grown plant samples, and field infected blueberry samples by restriction digestion of the rpoD gene PCR product (amplified with primers RST31 and RST33) using the BtsI enzyme. The LAMP assay allowed for specific real-time amplification of a 204-bp portion of the XfmrpoD gene from both pure bacterial cultures and infected plant material using the Genie® III system, a result further affirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. These detection strategies have the potential to greatly aid existing diagnostic methods for determining the distribution and prevalence of these Xf subspecies causing bacterial leaf scorch (BLS) in blueberries in the southeastern United States.

First Report of Citrus tristeza virus Infecting Citrus Trees in Georgia, USA.

Citrus tristeza virus (CTV) [genus Closterovirus; family Closteroviridae] is one of the most important, economically devastating viruses of citrus worldwide. On citrus trees grafted onto sour orange rootstock, typical CTV symptoms include dieback and defoliation, stunting, curling and chlorotic leaves, stem-pitting, and pinholes below the bud union on the inner face of the bark (Moreno et al. 2008). This single-stranded, positive-sense RNA virus is most efficiently transmitted by the brown citrus aphid (Toxoptera citricida), but it can also be transmitted by other aphid species and through grafting of infected plant material onto healthy plants (Moreno et al 2008; Herron et al. 2006). In Fall 2020, leaf material for virus testing was collected from 13 navel orange trees (Citrus × sinensis) grafted onto Poncirus trifoliata rootstocks (including 'Flying Dragon') located in a citrus research orchard in Tifton, GA. Trees ranged in age from 2 to 10 years, with the younger trees having been grafted from cuttings taken from the older trees. The oldest of these trees was derived from cuttings taken in 2009 from an orange tree growing locally in a residential yard in Tifton; this parent tree was more than 15 years old when these cuttings were obtained and was no longer available for sampling as of 2020. Symptoms or other visible signs of disease had not been noted on any of the tested trees, and trees were chosen for testing prior to the further dissemination of this plant material. The presence of CTV was verified via molecular and serological testing. CTV infection was initially confirmed in 8 of 13 tested samples using the ImmunoStrip® for CTV assay (Agdia® Inc., Elkhart, IN, cat no: ISK 78900/0025) according to the manufacturer's instructions. RNA was extracted from leaf material collected from the 13 sampled trees using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Following cDNA synthesis, samples were tested for the presence of CTV by reverse-transcription PCR using primer pair AR18F (5'-ATGTCAGGCAGCTTGGGAAATT-3') and AR18R (5'-TTCGTGTCTAAGTCRCGCTAAACA-3') which produces a 511 bp amplicon (Roy et al., 2005). PCR reactions confirmed the presence of CTV, with the same eight samples that had previously tested positive via Immunostrip® producing PCR fragments of the expected size. Amplified products from two of these samples were then sequenced using Sanger sequencing (Retrogen Inc, San Diego, CA, USA) and subjected to BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for further identification. Sequence analysis revealed that the obtained partial sequences (MW540805) from the p18 gene of both isolates were 100% identical to one another and shared 100% identity to corresponding sequences from CTV strain N4 (MK779711.1). To the best of our knowledge, this is the first report of CTV infecting citrus plants in Georgia. CTV could pose an imminent threat to the emerging citrus industry in Georgia if it were to become established in commercial citrus plantings either via the dissemination of infected plant material or via vector transfer of the virus under field conditions. While the brown citrus aphid is not known to be widespread in Georgia at this time, other CTV vectors are prevalent including the cotton aphid (Aphis gossypii) and the black citrus aphid (T. aurantia). Georgia citrus growers and plant propagators should be aware of this virus and take appropriate control measures to prevent the spread of this viral diseas.

Correction to: Genome-enabled insights into the biology of thrips as crop pests.

An amendment to this paper has been published and can be accessed via the original article.

Genome-enabled insights into the biology of thrips as crop pests.

BACKGROUND: The western flower thrips, Frankliniella occidentalis (Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present the F. occidentalis draft genome assembly and official gene set. RESULTS: We report on the first genome sequence for any member of the insect order Thysanoptera. Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly (size = 415.8 Mb, scaffold N50 = 948.9 kb) revealed a relatively complete and well-annotated assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, of which ~ 10% were manually verified and corrected by our consortium. We focused on manual annotation, phylogenetic, and expression evidence analyses for gene sets centered on primary themes in the life histories and activities of plant-colonizing insects. Highlights include the following: (1) divergent clades and large expansions in genes associated with environmental sensing (chemosensory receptors) and detoxification (CYP4, CYP6, and CCE enzymes) of substances encountered in agricultural environments; (2) a comprehensive set of salivary gland genes supported by enriched expression; (3) apparent absence of members of the IMD innate immune defense pathway; and (4) developmental- and sex-specific expression analyses of genes associated with progression from larvae to adulthood through neometaboly, a distinct form of maturation differing from either incomplete or complete metamorphosis in the Insecta. CONCLUSIONS: Analysis of the F. occidentalis genome offers insights into the polyphagous behavior of this insect pest that finds, colonizes, and survives on a widely diverse array of plants. The genomic resources presented here enable a more complete analysis of insect evolution and biology, providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers a genomic benchmark for molecular and evolutionary investigations of other Thysanoptera species.

First Report of Hop stunt viroid Infecting Citrus Trees in Georgia, USA.

In recent years, citrus production has rapidly increased within the state of Georgia (USA), and there are now citrus plantings within at least 32 counties in residential, production, and nursery settings. Among the pathogens capable of infecting citrus are viroids, the smallest plant pathogens. Viroids are comprised of circular, single-stranded RNA ranging from 246-463 nucleotides in length (Ito et al., 2002). Hop stunt viroid (HSVd) is one of several viroids known to infect citrus. This viroid has been previously reported within Arizona, California, Florida, Texas, and Washington in the United States and in other locations throughout the world (Hadidi, 2017). HSVd is often spread mechanically on contaminated tools or through grafting. With a wide host range that includes the families Moraceae, Rosaceae, and Rutaceae (citrus), this viroid can easily move throughout a nursery and spread to other plants (Hadidi, 2017). Symptoms of HSVd include a discoloration and gumming of phloem tissues, stem pitting, bark splitting, and chlorotic and stunted growth in susceptible citrus varieties including tangerines and their hybrids (Hadidi, 2017). There are not typically symptoms on leaves or fruits; however, lime plants have shown some yellowing on leaves (Hadidi, 2017). In May and June of 2020, leaf samples were collected from 12 different citrus plants in nursery settings in Berrien and Mitchell counties in Georgia. The cultivars sampled from Citrus reticulata 'Dekopon'. The sampled trees looked relatively healthy with little or no signs of damage, but were selected for testing to ensure that they were viroid free. Reverse transcription-polymerase chain reaction (RT-PCR) was initially used to verify infection with HSVd. Genomic RNA was extracted from the leaf tissue of twelve plants using the TRIzol reagent (Thermofisher, Waltham, MA). Following cDNA synthesis, samples were tested for the presence of HSVd using the primer pair HSVd-F (5'-GGCAACTCTTCTCAGAATCCAGC-3') and HSVd-R (5'-CCGGGGCTCCTTTCTCAGGTAAGT-3') which produces a 302 bp amplicon (Sano et al., 1988). The PCR reactions for nine of the tested samples did not result in the production of any bands, however the other three samples, all Citrus reticulata 'Dekopon', produced the expected amplicon for HSVd. The amplified products were sequenced using Sanger sequencing (Retrogen Inc, San Diego, CA, USA) and the identity of the fragment sequences was confirmed using BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Partial sequences from these amplicons (deposited as accession number MT632007) shared 99% identity to corresponding HSVd sequences in Genbank (accession number MG779542). In addition to RT-PCR and sequencing, the recombinase-polymerase-amplification (RPA) technology based AmplifyRP® Acceler8™ end-point detection assay (Agdia® Inc., Elkhart, IN) was performed on previously confirmed tissue according to the manufacturer's instructions. This assay also confirmed the presence of HSVd viroid in the three samples that had been previously confirmed via RT-PCR. To the best of our knowledge, this is the first report of HSVd infecting Citrus reticulata 'Dekopon' in Georgia. If this viroid were to spread within the growing Georgia citrus industry, it could pose a significant threat to citrus plantings that contain susceptible varieties. Nursery stock infected with this viroid should be destroyed, and Georgia nursery producers and citrus growers should take appropriate precautions to prevent the spread of this viroid disease, including properly sanitizing tools used for citrus grafting and pruning. Further research is needed to determine the distribution of HSVd and its potential to impact commercial citrus production in Georgia.

Natural Infection of Southern Highbush Blueberry (Vaccinium corymbosum Interspecific Hybrids) by Xylella fastidiosa subsp. fastidiosa.

Xylella fastidiosa (Xf) is an emerging insect-vectored, xylem-limited bacterium that can cause disease on several economically important fruit and tree crops including almond, blueberry, citrus, grapevine, peach, and pecan. On blueberry, Xf causes bacterial leaf scorch (BLS), which is prevalent in the southeastern United States. This disease, previously reported to be caused by Xf subsp. multiplex (Xfm), can result in rapid plant decline and death of southern highbush (SHB) blueberry cultivars. In 2017, a survey of blueberry plantings in southern Georgia (U.S.A.) confirmed the presence of Xf-infected plants in eight of nine sites examined, and seven isolates were cultured from infected plants. Genetic characterization of these isolates through single-locus and multilocus sequence analysis revealed that three isolates from two sites belonged to Xf subsp. fastidiosa (Xff), with significant similarity to isolates from grapevine. After these three isolates were artificially inoculated onto greenhouse-grown SHB blueberries (cv. 'Rebel'), symptoms typical of BLS developed, and Xff infection was confirmed through genetic characterization and reisolation of the bacterium to fulfill Koch's postulates. Because all previously reported Xf isolates from blueberry have been characterized as Xfm, this is the first time that isolation of Xff has been reported from naturally infected blueberry plantings. The potential impact of Xff isolates on disease management in blueberry requires further exploration. Furthermore, given that isolates from both Xfm and Xff were obtained within a single naturally infected blueberry planting, blueberry in southern Georgia may provide opportunities for intersubspecific recombination between Xff and Xfm isolates.

Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus.

A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie® III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.

Comparative analysis of different molecular and serological methods for detection of Xylella fastidiosa in blueberry.

Bacterial leaf scorch, caused by Xylella fastidiosa, is a major threat to blueberry production in the southeastern United States. Management of this devastating disease is challenging and often requires early detection of the pathogen to reduce major loss. There are several different molecular and serological detection methods available to identify the pathogen. Knowing the efficiency and suitability of these detection techniques for application in both field and laboratory conditions is important when selecting the appropriate detection tool. Here, we compared the efficiency and the functionality of four different molecular detection techniques (PCR, real-time PCR, LAMP and AmplifyRP® Acceler8™) and one serological detection technique (DAS-ELISA). The most sensitive method was found to be real-time PCR with the detection limit of 25 fg of DNA molecules per reaction (≈9 genome copies), followed by LAMP at 250 fg per reaction (≈90 copies), AmplifyRP® Acceler8™ at 1 pg per reaction (≈350 copies), conventional PCR with nearly 1.25 pg per reaction (≈ 440 copies) and DAS-ELISA with 1x105 cfu/mL of Xylella fastidiosa. Validation between assays with 10 experimental samples gave consistent results beyond the variation of the detection limit. Considering robustness, portability, and cost, LAMP and AmplifyRP® Acceler8™ were not only the fastest methods but also portable to the field and didn't require any skilled labor to carry out. Among those two, AmplifyRP® Acceler8™ was faster but more expensive and less sensitive than LAMP. On the other hand, real-time PCR was the most sensitive assay and required comparatively lesser time than C-PCR and DAS-ELISA, which were the least sensitive assays in this study, but all three assays are not portable and needed skilled labor to proceed. These findings should enable growers, agents, and diagnosticians to make informed decisions regarding the selection of an appropriate diagnostic tool for X. fastidiosa on blueberry.

The bacterial pathogen Xylella fastidiosa affects the leaf ionome of plant hosts during infection.

Xylella fastidiosa is a plant pathogenic bacterium that lives inside the host xylem vessels, where it forms biofilm believed to be responsible for disrupting the passage of water and nutrients. Here, Nicotiana tabacum was infected with X. fastidiosa, and the spatial and temporal changes in the whole-leaf ionome (i.e. the mineral and trace element composition) were measured as the host plant transitioned from healthy to diseased physiological status. The elemental composition of leaves was used as an indicator of the physiological changes in the host at a specific time and relative position during plant development. Bacterial infection was found to cause significant increases in concentrations of calcium prior to the appearance of symptoms and decreases in concentrations of phosphorous after symptoms appeared. Field-collected leaves from multiple varieties of grape, blueberry, and pecan plants grown in different locations over a four-year period in the Southeastern US showed the same alterations in Ca and P. This descriptive ionomics approach characterizes the existence of a mineral element-based response to X. fastidiosa using a model system suitable for further manipulation to uncover additional details of the role of mineral elements during plant-pathogen interactions. This is the first report on the dynamics of changes in the ionome of the host plant throughout the process of infection by a pathogen.