Diversity of 'Candidatus Liberibacter asiaticus' Strains in Texas Revealed by Prophage Sequence Analyses.

Prophages/phages are important components of the genome of 'Candidatus Liberibacter asiaticus' (CLas), an unculturable alphaproteobacterium associated with citrus huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida but different to those reported in California and China. Further studies were performed on prophage type diversity. Analyses on Type 1-specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originating from Florida, and Type 1-B, clustered with prophage P-SGCA5-1 originating in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2-specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. The results from this study provide an initial baseline of information on genomic and population diversity of CLas in Texas.

A Field Deployable Real-Time Loop-Mediated Isothermal Amplification Targeting Five Copy nrdB Gene for the Detection of 'Candidatus Liberibacter asiaticus' in Citrus.

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, 'Candidatus Liberibacter asiaticus' (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, Diaphorina citri Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy nrdB gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the nrdB target as low as ~2.6 Log10 copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.

Co-Occurrence Analysis of Citrus Root Bacterial Microbiota under Citrus Greening Disease.

Candidatus Liberibacter asiaticus (CLas) is associated with Citrus Huanglongbing (HLB), a devastating disease in the US. Previously, we conducted a two-year-long monthly HLB survey by quantitative real-time PCR using root DNA fractions prepared from 112 field grapefruit trees grafted on sour orange rootstock. Approximately 10% of the trees remained CLas-free during the entire survey period. This study conducted 16S metagenomics using the time-series root DNA fractions, monthly prepared during twenty-four consecutive months, followed by microbial co-occurrence network analysis to investigate the microbial factors contributing to the CLas-free phenotype of the aforementioned trees. Based on the HLB status and the time when the trees were first diagnosed as CLas-positive during the survey, the samples were divided into four groups, Stage H (healthy), Stage I (early), II (mid), and III (late) samples. The 16S metagenomics data using Silva 16S database v132 revealed that HLB compromised the diversity of rhizosphere microbiota. At the phylum level, Actinobacteria and Proteobacteria were the predominant bacterial phyla, comprising >93% of total bacterial phyla, irrespective of HLB status. In addition, a temporal change in the rhizosphere microbe population was observed during a two-year-long survey, from which we confirmed that some bacterial families differently responded to HLB disease status. The clustering of the bacterial co-occurrence network data revealed the presence of a subnetwork composed of Streptomycetaceae and bacterial families with plant growth-promoting activity in Stage H and III samples. These data implicated that the Streptomycetaceae subnetwork may act as a functional unit against HLB.

Host-derived chimeric peptides clear the causative bacteria and augment host innate immunity during infection: A case study of HLB in citrus and fire blight in apple.

Bacterial diseases cause severe losses in the production and revenue of many fruit crops, including citrus and apple. Huanglongbing (HLB) in citrus and fire blight in apple are two deadly diseases without any cure. In this article, we introduce a novel therapy for HLB and fire blight by enhancing the innate immunity of the host plants. Specifically, we constructed in silico a library of chimeras containing two different host peptides with observed or predicted antibacterial activity. Subsequently, we performed bactericidal and toxicity tests in vitro to select a few non-toxic chimeras with high antibacterial activity. Finally, we conducted ex planta studies to show that not only do the chimeras clear the causative bacteria from citrus leaves with HLB and from apple leaves with fire blight but they also augment the host's innate immunity during infection. This platform technology can be extended to design host-derived chimeras against multiple pathogenic bacteria that cause diseases in plants and animals of agricultural importance and in humans.

Diversity of Citrus tristeza virus Strains in the Upper Gulf Coast Area of Texas.

Citrus tristeza virus (CTV) in Texas was first reported in the 1950s and has since been sporadically reported in the residential areas in the Upper Gulf Coast region. Because the major rootstock for commercial citriculture in South Texas is sour orange, which is susceptible to CTV decline, the spread of CTV into South Texas can pose a great threat to Texas citrus industry. Thirty-six CTV-positive samples, collected during surveys conducted in the Upper Gulf Coast area of Texas from 2013 to 2018, were first analyzed by strain-specific real-time PCR (RT-PCR) targeting various regions of CTV Open reading frame (Orf) 1a and then by amplicon sequencing derived from p25 and p20 region of CTV genome. Among 36 samples, 33 were successfully genotyped by strain-specific RT-PCR and by amplicon sequencing followed by phylogenetic analysis. Variability in the detection of CTV strains was observed over a 6-year period. In 2013, T3 and T30 were the only strains detected in the Upper Gulf Coast of Texas, but in further surveys until 2018, additional strains were detected, including T36, VT, and RB. Mixed infections were also detected in 14 samples comprising about 42% of CTV samples examined in the study. Although genotyping mixed infection samples by targeting Orf 1a and full-length p25, residing in the 5' and 3' region of the CTV genome, respectively, confirmed the presence of multiple strains in these samples, incongruent genotyping data were observed. These findings suggested that the current status of CTV strain diversity in Texas Upper Gulf Coast region might have been established by multiple introductions of CTV-infected plant materials for propagation and with a potential recombination in planta.

Root samples provide early and improved detection of Candidatus Liberibacter asiaticus in Citrus.

Huanglongbing (HLB), or Citrus Greening, is one of the most devastating diseases affecting agriculture today. Widespread throughout Citrus growing regions of the world, it has had severe economic consequences in all areas it has invaded. With no treatment available, management strategies focus on suppression and containment. Effective use of these costly control strategies relies on rapid and accurate identification of infected plants. Unfortunately, symptoms of the disease are slow to develop and indistinct from symptoms of other biotic/abiotic stressors. As a result, diagnosticians have focused on detecting the pathogen, Candidatus Liberibacter asiaticus, by DNA-based detection strategies utilizing leaf midribs for sampling. Recent work has shown that fibrous root decline occurs in HLB-affected trees before symptom development among leaves. Moreover, the pathogen, Ca. Liberibacter asiaticus, has been shown to be more evenly distributed within roots than within the canopy. Motivated by these observations, a longitudinal study of young asymptomatic trees was established to observe the spread of disease through time and test the relative effectiveness of leaf- and root-based detection strategies. Detection of the pathogen occurred earlier, more consistently, and more often in root samples than in leaf samples. Moreover, little influence of geography or host variety was found on the probability of detection.

Incidence, Severity, and Characterization of Phytophthora Foot Rot of Citrus in Texas and Implications for Disease Management.

Phytophthora-induced foot rot, also known as gummosis, is an important disease affecting citrus production worldwide. In Texas, the third-largest citrus-producing state in the United States, limited information is available on the etiology and epidemiology of foot rot in commercial orchards. This study comprises a survey of foot rot incidence and severity in Texas and the characterization of Phytophthora isolates associated with the disease. Surveys in 2015 and 2017 of 30 orchards in the Lower Rio Grande Valley (LRGV) region where commercial citrus production is concentrated in the state revealed that foot rot occurred in 97% of the orchards assessed. Overall, foot rot symptoms were observed on 33.7% of the trees evaluated and the disease severity index in the region was rated at 14.2 and 16.5% in 2015 and 2017, respectively. Lesions were mostly present on the scion, while the rootstock (sour orange) was not affected. Phytophthora nicotianae was the only Phytophthora sp. isolated from the surveyed orchards and from five additional residential sites on the Texas Coastal Bend (TCB). Sporangia and chlamydospores from 34 representative LRGV isolates of P. nicotianae were larger than those of TCB isolates. In both LRGV and TCB, A1 and A2 mating types were present in the same location, albeit the A2 mating type was more prevalent. All isolates were sensitive to mefenoxam (50% inhibition in the presence of mefenoxam [EC50] < 0.5 µg/ml), except for one TCB isolate (EC50 = 143.6 µg/ml). Our research indicates that treatment for Phytophthora foot rot in the region is necessary and, although mefenoxam is still useful, alternating chemistries for resistance management are required.

First report of the occurrence of Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae), an invasive species in Nigeria, West Africa.

The Asian citrus psyllid (ACP; Diaphorina citri) is the vector of Candidatus Liberibacter asiaticus (CLas) that is associated with the devastating Huanglongbing (HLB; citrus greening disease). This pest of Asian origin has spread into the Americas and more recently into a few countries in East Africa. During recent surveys, suspect ACP adults and nymphs were recorded for the first time infesting citrus trees in southwest Nigeria. Morphological identification and DNA barcoding confirmed the samples to be D. citri. Analysis of the obtained sequences revealed that the ACP recorded in Nigeria clustered with other taxa in the previously identified B1 clade that consists of populations from different continents. The presence of the endosymbionts Ca. Carsonella ruddii and Ca. Profftella armatura in ACP from Nigeria was also confirmed by PCR and Sanger sequencing. The ACP individuals were assayed for the presence of CLaf, CLam and CLas by qPCR, but none of the insects tested positive for any of the Liberibacters. The prolific nature of ACP and the tropical climate prevailing in the citrus-producing areas of Nigeria and other West African countries may favor its rapid spread and population increase, thus posing a grave threat to the sustainability of citriculture in these countries.

Distribution of Candidatus Liberibacter asiaticus in Citrus and the Asian Citrus Psyllid in Texas Over a Decade.

Huanglongbing (HLB, citrus greening disease) in the major citrus-producing states of the United States is associated with Candidatus Liberibacter asiaticus (CLas), which is vectored by the Asian citrus psyllid (ACP). Surveys were conducted in Texas from 2007 to 2017 to assess the prevalence and titer of CLas in ACPs and citrus trees. ACP and citrus leaf tissue samples were collected from suspect trees in residential areas and commercial groves (orchards) and assayed for CLas by quantitative PCR. CLas detection in ACPs (2011) preceded that of citrus trees (2012) by several months. Annual incidences of CLas-positive ACPs and leaf tissue followed an exponential growth pattern over the survey period, varying from 0.03 to 28.7% in ACPs and 0.6 to 36.5% in citrus trees. There was a significant and positive relationship between the monthly incidences of CLas-positive ACP and leaf tissue samples. The proportion of HLB detection sites also increased with time, reaching 26 and 40% of commercial groves and residential sites, respectively, by 2017. Seasonal variations were observed in the incidences of CLas-positive ACPs and citrus trees such that significantly more CLas-positive ACPs and trees were recorded during the fall and winter of a given year relative to the hot summer. A temporal analysis of the class distribution of cycle threshold values revealed a trend of increased bacterial accumulation in ACPs and trees over time, with the trend more pronounced for the former than the latter host type. These findings provide a comprehensive insight into the ongoing CLas/HLB epidemic in Texas, with potential lessons for California and other citrus-producing areas where the disease is not yet established.

Pathogenicity of Zygosaccharomyces bailii and Other Yeast Species to Mexican Fruit Fly (Diptera: Tephritidae) and Mass Rearing Implications.

Yeasts from all immature life stages of Mexican fruit fly Anastrepha ludens (Loew) (Diptera: Tephritidae) from diet, insectary air, and rearing materials were isolated, identified and evaluated for pathogenicity. Fifteen species of yeasts with one to genus level were identified from 72 yeast cultures obtained. Zygosaccharomyces bailii was the only yeast found to be highly pathogenic to Mexican fruit fly. Seventy-two hours post inoculation, the diet in bioassay cups with Z. bailii consistently showed signs of fermentation with gas bubbling causing the migration of larvae to the walls and lids of bioassay cups. The spent diet from Z. balii-infested cups was crusty, cracked and had a pasty layer. Many larvae were small, moribund, and discolored, appearing caramel or blackish. Insect yield loss with Z. bailii in comparison to that of control ranged from 10 to 44% for larvae and 14 to 47% for pupae. Additionally, Z. bailii caused a reduction in mean pupal weight. The weakly pathogenic yeasts produced significantly less yield of larvae and pupae than the nonpathogenic ones included Trichosporon montevideense, Clavispora lucitaniae, Candida sp., C. rugosa, and Rhodotorula mucilaginosa. Yield loss of larvae caused by this group ranged from 12 (C. lusitaniae) to 18% (R. mucilaginosa). Yield losses for pupa were similar to that of larvae. The mean pupa weight for these species was above the minimum acceptable (16.50 mg) for the SIT program. The nonpathogenic yeast produced yields of larvae and pupae similar to the control included Cryptococcus diffluens, Pichia kudriavzevii, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Trichosporon asahii, Debaryomyces hansenii, Candida tropicalis, Cryptococcus sp., Candida parapsilosis, and Hanseniaspora opuntiae. In conclusion, the identification and management of insect pathogenic yeasts, such as Z. balii in mass rearing systems of Mexican fruit fly must be considered to avoid their potential negative effects.

Draft Whole-Genome Sequence of "Candidatus Liberibacter asiaticus" Strain TX2351 Isolated from Asian Citrus Psyllids in Texas, USA.

We report here the draft genome sequence of "Candidatus Liberibacter asiaticus" strain TX2351, collected from Asian citrus psyllids in south Texas, USA. The TX2351 genome has a size of 1,252,043 bp, a G+C content of 36.5%, 1,184 predicted open reading frames, and 52 RNA genes.

Enhanced Acquisition Rates of 'Candidatus Liberibacter asiaticus' by the Asian Citrus Psyllid (Hemiptera: Liviidae) in the Presence of Vegetative Flush Growth in Citrus.

The Asian citrus psyllid preferentially feeds and exclusively reproduces on young, newly emerged flush shoots of citrus. Asian citrus psyllid nymphs feed and complete their life stages on these flush shoots. Recent studies conducted under greenhouse conditions have shown that the transmission rates of 'Candidatus Liberibacter asiaticus' (CLas), the putative causal agent of huanglongbing disease of citrus, are enhanced when flush shoots are present. However, it is unclear if CLas acquisition by migrant adult Asian citrus psyllids is similarly enhanced. To address this knowledge gap, cohorts of Asian citrus psyllid adults were allowed 1-wk acquisition access period (AAP) on flushing and nonflushing shoots of qPCR-tested symptomatic (CLas+) and asymptomatic (CLas-) 10-yr-old sweet orange trees under field conditions. After the AAP, they were tested for CLas by qPCR. Progeny Asian citrus psyllid adults that emerged 4 wk post-AAP were similarly retrieved and tested. Eighty percent of flushing and 30% of nonflushing CLas+ trees produced infective Asian citrus psyllid adults, indicating that flush shoots have greater potential to be inoculum sources for CLas acquisition. Concomitantly, 21.1% and 6.0% infective adults were retrieved, respectively, from flushing and nonflushing CLas+ trees, indicating that Asian citrus psyllid adults acquire CLas more efficiently from flush shoots relative to mature shoots. In addition, 12.1% of infective Asian citrus psyllid adult progeny were obtained from 70% of flushing CLas+ trees. Significantly lower mean Ct values were also obtained from infective adults retrieved from flushing relative to nonflushing trees. The results underscore the role of flush shoots in CLas acquisition and the need to protect citrus trees from Asian citrus psyllid infestations during flush cycles.

Distribution of 'Candidatus Liberibacter asiaticus' Above and Below Ground in Texas Citrus.

Detection of 'Candidatus Liberibacter asiaticus' represents one of the most difficult, yet critical, steps of controlling Huanglongbing disease. Efficient detection relies on understanding the underlying distribution of bacteria within trees. To that end, we studied the distribution of 'Ca. L. asiaticus' in leaves of 'Rio Red' grapefruit trees and in roots of 'Valencia' sweet orange trees grafted onto sour orange rootstock. We performed two sets of leaf collection on grapefruit trees; the first a selective sampling targeting symptomatic leaves and their neighbors and the second a systematic collection disregarding symptomology. From uprooted orange trees, we exhaustively sampled fibrous roots. In this study, the presence of 'Ca. L. asiaticus' was detected in leaves using real-time polymerase chain reaction (PCR) targeting the 16S ribosomal gene and in roots using the rpIJ/rpIL ribosomal protein genes and was confirmed with conventional PCR and sequencing of the rpIJ/rpIL gene in both tissues. Among randomly collected leaves, 'Ca. L. asiaticus' was distributed in a patchy fashion. Detection of 'Ca. L. asiaticus' varied with leaf symptomology with symptomatic leaves showing the highest frequency (74%) followed by their neighboring asymptomatic leaves (30%), while randomly distributed asymptomatic leaves had the lowest frequency (20%). Among symptomatic leaves, we found statistically significant differences in mean number of bacterial cells with respect to both increasing distance of the leaf from the trunk and cardinal direction. The titer of 'Ca. L. asiaticus' cells was significantly greater on the north side of trees than on the south and west sides. Moreover, these directions showed different spatial distributions of 'Ca. L. asiaticus' with higher titers near the trunk on the south and west sides as opposed to further from the trunk on the north side. Similarly, we found spatial variation in 'Ca. L. asiaticus' distribution among root samples. 'Ca. L. asiaticus' was detected more frequently and bacterial abundances were higher among horizontally growing roots just under the soil surface (96%) than among deeper vertically growing roots (78%). Bacterial abundance declined slightly with distance from the trunk. These results point to paths of research that will likely prove useful to combating this devastating disease.

Silverleaf Nightshade (Solanum elaeagnifolium), a Reservoir Host for 'Candidatus Liberibacter solanacearum', the Putative Causal Agent of Zebra Chip Disease of Potato.

Zebra chip disease of potato is caused by the bacterial pathogen 'Candidatus Liberibacter solanacearum' and is a growing concern for commercial potato production in several countries in North and Central America and New Zealand. 'Ca. L. solanacearum' is vectored by the potato psyllid Bactericera cockerelli, which transmits the pathogen to several cultivated and wild solanaceaous host plants. Silverleaf nightshade (SLN), Solanum elaeagnifolium, is a common weed in the Lower Rio Grande Valley of Texas and a host for both the potato psyllid and 'Ca. L. solanacearum'. SLN plants were successfully inoculated with 'Ca. L. solanacearum' under laboratory conditions. Retention studies demonstrated that 'Ca. L. solanacearum'-infected SLN planted in the field in January 2013, concurrent with commercial potato planting, retained the pathogen under field conditions throughout the year despite extensive dieback during summer. The presence of 'Ca. L. solanacearum' was confirmed in leaves, roots, and stolons of SLN plants collected the following year using polymerase chain reaction. Acquisition assays using B. cockerelli adults also revealed that SLN retained the pathogen. Transmission studies determined that B. cockerelli can acquire 'Ca. L. solanacearum' within a 2-week acquisition access period on 'Ca. L. solanacearum'-infected SLN and subsequently transmit the pathogen to potato. These results demonstrate that SLN plants can serve as a reservoir for 'Ca. L. solanacearum', providing a source of inoculum for B. cockerelli adults colonizing potato the next season. The presence of SLN plants all year round in the LRGV makes the weed an epidemiologically important host. These findings underscore the importance of eradicating or managing SLN plants growing in the vicinity of potato fields to prevent spread of 'Ca. L. solanacearum' and damage caused by zebra chip.

Changes in free amino acids and polyamine levels in Satsuma leaves in response to Asian citrus psyllid infestation and water stress.

The effects of biotic and abiotic stresses on changes in amino acids and polyamine levels in Satsuma orange (Citrus unshiu; cultivar Owari) leaves were investigated. Asian citrus psyllids Diaphorina citri (Kuwayama) (ACP) infestation was used to induce biotic stress while a water deficit was imposed to induce abiotic stress. Potted trees were infested by placing 50 psyllids on 3 citrus leaves enclosed in nylon mesh bags for 5 d. A parallel set of plants were kept water stressed by maintaining the soil at 20% water holding capacity for 5 d. Levels of total free amino acids were higher in water stressed and ACP infested leaves. Polyamine putrescine increased in infested leaves but not in water stressed leaves. Proline was the most abundant amino acid and its levels significantly increased by both biotic and abiotic stresses. Proline levels in infested leaves were significantly higher than the water stressed leaves. Histidine, methionine, asparagine, arginine, serine, and leucine levels also increased significantly in infested leaves, but in water stressed leaves only leucine, methionine, and threonine increased. Levels of amino acids, such as tyrosine, isoleucine, phenylalanine, glutamic acid, and alanine, declined in infested leaves. Under water stress asparagine, phenylalanine, serine, and histidine also declined compared to controls. This indicates that while proteolysis occurred under both stresses, metabolic conversion of amino acids was different under the two stresses. In ACP infested leaves some amino acids may be used as feeding material and/or converted into secondary metabolites for defense.

Functional dependence on calcineurin by variants of the Saccharomyces cerevisiae vacuolar Ca2+/H+ exchanger Vcx1p.

The Ca(2+)-dependent protein phosphatase calcineurin is an important regulator of ion transporters from many organisms, including the Saccharomyces cerevisiae vacuolar Ca(2+)/H(+) exchanger Vcx1p. In yeast and plants, cation/H(+) exchangers are important in shaping cytosolic Ca(2+) levels involved in signal transduction and providing tolerance to potentially toxic concentrations of cations such as Ca(2+), Mn(2+) and Cd(2+). Previous genetic evidence suggested Vcx1p is negatively regulated by calcineurin. By utilizing direct transport measurements into vacuolar membrane vesicles, we demonstrate that Vcx1p is a low-affinity Ca(2+) transporter and may also function in Cd(2+) transport, but cannot transport Mn(2+). Furthermore, direct Ca(2+) transport by Vcx1p is calcineurin sensitive. Using a yeast growth assay, a mutant allele of VCX1 (VCX1-S204A/L208P), termed VCX1-M1, was previously found to confer strong Mn(2+) tolerance. Here we demonstrate that this Mn(2+) tolerance is independent of the Ca(2+)/Mn(2+)-ATPase Pmr1p and results from Mn(2+)-specific vacuolar transport activity of Vcx1-M1p. This Mn(2+) transport by Vcx1-M1p is calcineurin dependent, although the localization of Vcx1-M1p to the vacuole appears to be calcineurin independent. Additionally, we demonstrate that mutation of L208P alone is enough to confer calcineurin-dependent Mn(2+) tolerance. This study demonstrates that calcineurin can positively regulate the transport of cations by VCX1-M1p.